HCV protease inhibitors and uses thereof

ABSTRACT

The present invention provides compounds, pharmaceutically acceptable compositions thereof, and methods of using the same.

CROSS REFERENCE TO RELATED APPLICATIONS

The present invention claims priority to U.S. provisional applicationSer. No. 61/089,269, filed Aug. 15, 2008, and U.S. provisionalapplication Ser. No. 61,098,662, filed Sep. 19, 2008, the entirety ofeach of which is hereby incorporated by reference.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to compounds useful as inhibitors of HCVprotease. The invention also provides pharmaceutically acceptablecompositions comprising compounds of the present invention and methodsof using said compositions in the treatment of various disorders.

In accordance with 37 CFR 1.52(e)(5), a Sequence Listing in the form ofa text file (entitled “Sequence Listing.txt,” created on Sept. 28, 2009,and 19 kilobytes) is incorporated herein by reference in its entirety.cl BACKGROUND OF THE INVENTION

It is estimated that over 170 million people worldwide are infected withthe Hepatitis C virus (HCV). With an estimated human sero-prevalence of3% globally, HCV is the major cause for most cases of non-A, non-Bhepatitis, (Alberti, A. et al., J. Hepatology 31, (Suppl. 1): 17-24,1999). While the symptoms of acute hepatitis subside in some patients,at least 85% of HCV infections become chronic, and 20% of those infecteddevelop liver cirrhosis. There is less than a 50% survival rate at fouryears post cirrhosis diagnosis. Chronic HCV infection is also associatedwith increased incidence of hepatocellular carcinoma.

HCV is a positive-stranded RNA virus whose genome encodes a polyproteinof approximately 3000 amino acids. This precursor protein is processedinto at least 10 viral structural and nonstructural proteins: C, E1, E2,p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B (Blight, K. J., et al.,Antiviral Ther. 3, Suppl. 3: 71-81, 1998). HCV nonstructural (NS)proteins are derived by proteolytic cleavage of the polyprotein and arepresumed to provide the essential catalytic machinery for viralreplication.

NS3 is an approximately 68 Kda protein, and has both an N-terminalserine protease domain and an RNA-dependent ATPase domain at itsC-terminus. It has been shown that the NS4A protein serves as aco-factor for the serine protease activity of NS3. NS3 functions as aproteolytic enzyme that cleaves sites liberating other nonstructuralproteins necessary for HCV replication and is a viable therapeutictarget for antiviral chemotherapy.

No vaccines are available for HCV, and the established therapy ofinterferon treatment is effective in only 15-20% of patients (Weiland,O., FEMS Microbiol. Rev. 14: 279-88, 1994), and has significant sideeffects (Walker, M. A., et al., DDT 4: 518-29, 1999; Moradpour, D., etal., Eur. J. Gastroenterol. Hepatol. 11: 1199-1202, 1999). While thecurrent standard of care, pegylated interferon a in combination withribavirin, is more efficacious and appears to decrease hepatocellularcarcinoma in patients with HCV-related cirrhosis (Hung, C. H., et al., JViral Hepatitis 13(6): 409-414, 2006), this treatment has also beenshown to produce side effects such as thyroid dysfunction (Huang, J. F.,et al., J Viral Hepatitis 13(6): 396-401, 2006).

The poor prognosis for patients suffering from HCV infection and thecurrent lack of effective, approved treatments, highlights theoverwhelming need for new inhibitors of HCV NS3 protease.

SUMMARY OF THE INVENTION

It has now been found that compounds of this invention, andpharmaceutically acceptable compositions thereof, are effective asinhibitors of HCV protease. Such compounds have the general formula I:

or a pharmaceutically acceptable salt thereof, wherein R¹, R², R³, R⁴,R^(a), R^(b), R^(x), and R^(y) are as defined herein.

Compounds of the present invention, and pharmaceutically acceptablecompositions thereof, are useful for treating a variety of diseases,disorders or conditions, associated with HCV. Such diseases, disorders,or conditions include those described herein.

Compounds provided by this invention are also useful for the study ofHCV protease in biological and pathological phenomena; the study ofintracellular signal transduction pathways mediated by HCV protease; andthe comparative evaluation of new HCV protease inhibitors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a mass spectroscopic analysis of HCV NS3/4A wild-typeprotease alone and in the presence of test compound I-1 (upper panel),and HCV NS3/4A mutant C159S protease in the presence of test compoundI-1 (lower panel).

FIG. 2 depicts a mass spectroscopic analysis of HCV NS3/4A wild-typeprotease in the presence of test compound I-11.

FIG. 3 depicts a mass spectroscopic analysis of HCV NS3/4A wild-typeprotease alone (upper panel) and in the presence of telaprevir (lowerpanel).

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

1. General Description of Compounds of the Invention:

In certain embodiments, the present invention provides a compound offormula I:

or a pharmaceutically acceptable salt thereof, wherein:

-   one of R^(a) and R^(b) is hydrogen and the other is —OH or —OC(O)R′,    or R^(a) and R^(b) are taken together to form an oxo group;-   R′ is an optionally substituted group selected from C₁₋₆ aliphatic,    C₃₋₇ cycloalkyl, 6-10 membered aryl, 5-10 membered heteroaryl having    1-4 heteroatoms independently selected from nitrogen, oxygen, or    sulfur, or 4-7 membered heterocyclyl having 1-2 heteroatoms    independently selected from nitrogen, oxygen, or sulfur;-   R^(x) and R^(y) are taken together to form an optionally substituted    C₃₋₇ membered ring having 0-2 heteroatoms independently selected    from nitrogen, oxygen, or sulfur;-   R¹ is an optionally substituted group selected from C₁₋₆ aliphatic    or C₃₋₇ cycloalkyl(C₁₋₃ alkyl);-   R² is hydrogen or an optionally substituted group selected from C₁₋₆    aliphatic or C₃₋₇ cycloalkyl;-   R³ is a warhead group;-   R⁴ is —NHC(O)NHR⁵, —NHC(O)OR⁶, or

-   R⁵ is an optionally substituted group selected from C₁₋₆ aliphatic,    bridged bicyclic, 6-10 membered aryl, 5-10 membered heteroaryl    having 1-4 heteroatoms independently selected from nitrogen, oxygen,    or sulfur, or 4-7 membered heterocyclyl having 1-2 heteroatoms    independently selected from nitrogen, oxygen, or sulfur;-   R⁶ is an optionally substituted group selected from C₁₋₆ aliphatic,    bridged bicyclic, 6-10 membered aryl, 5-10 membered heteroaryl    having 1-4 heteroatoms independently selected from nitrogen, oxygen,    or sulfur, or 4-7 membered heterocyclyl having 1-2 heteroatoms    independently selected from nitrogen, oxygen, or sulfur;-   R⁷ is an optionally substituted group selected from C₁₋₆ aliphatic,    bridged bicyclic, 6-10 membered aryl, 5-10 membered heteroaryl    having 1-4 heteroatoms independently selected from nitrogen, oxygen,    or sulfur, or 4-7 membered heterocyclyl having 1-2 heteroatoms    independently selected from nitrogen, oxygen, or sulfur;-   R is hydrogen or an optionally substituted group selected from C₁₋₆    aliphatic, C₃₋₇ cycloalkyl, 6-10 membered aryl, 5-10 membered    heteroaryl having 1-4 heteroatoms independently selected from    nitrogen, oxygen, or sulfur, or 4-7 membered heterocyclyl having 1-2    heteroatoms independently selected from nitrogen, oxygen, or sulfur.    2. Compounds and Definitions:

Compounds of this invention include those described generally above, andare further illustrated by the classes, subclasses, and speciesdisclosed herein. As used herein, the following definitions shall applyunless otherwise indicated. For purposes of this invention, the chemicalelements are identified in accordance with the Periodic Table of theElements, CAS version, Handbook of Chemistry and Physics, 75^(th) Ed.Additionally, general principles of organic chemistry are described in“Organic Chemistry”, Thomas Sorrell, University Science Books,Sausalito: 1999, and “March's Advanced Organic Chemistry”, 5^(th) Ed.,Ed.: Smith, M. B. and March, J., John Wiley & Sons, New York: 2001, theentire contents of which are hereby incorporated by reference.

The term “aliphatic” or “aliphatic group”, as used herein, means astraight-chain (i.e., unbranched) or branched, substituted orunsubstituted hydrocarbon chain that is completely saturated or thatcontains one or more units of unsaturation, or a monocyclic hydrocarbonor bicyclic hydrocarbon that is completely saturated or that containsone or more units of unsaturation, but which is not aromatic (alsoreferred to herein as “carbocycle,” “cycloaliphatic” or “cycloalkyl”),that has a single point of attachment to the rest of the molecule.Unless otherwise specified, aliphatic groups contain 1-6 aliphaticcarbon atoms. In some embodiments, aliphatic groups contain 1-5aliphatic carbon atoms. In other embodiments, aliphatic groups contain1-4 aliphatic carbon atoms. In still other embodiments, aliphatic groupscontain 1-3 aliphatic carbon atoms, and in yet other embodiments,aliphatic groups contain 1-2 aliphatic carbon atoms. In someembodiments, “cycloaliphatic” (or “carbocycle” or “cycloalkyl”) refersto a monocyclic C₃-C₆ hydrocarbon that is completely saturated or thatcontains one or more units of unsaturation, but which is not aromatic,that has a single point of attachment to the rest of the molecule.Suitable aliphatic groups include, but are not limited to, linear orbranched, substituted or unsubstituted alkyl, alkenyl, alkynyl groupsand hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or(cycloalkyl)alkenyl.

As used herein, the term “bridged bicyclic” refers to any bicyclic ringsystem, i.e. carbocyclic or heterocyclic, saturated or partiallyunsaturated, having at least one bridge. As defined by IUPAC, a “bridge”is an unbranched chain of atoms or an atom or a valence bond connectingtwo bridgeheads, where a “bridgehead” is any skeletal atom of the ringsystem which is bonded to three or more skeletal atoms (excludinghydrogen). In some embodiments, a bridged bicyclic group has 7-12 ringmembers and 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur. Such bridged bicyclic groups are well known in theart and include those groups set forth below where each group isattached to the rest of the molecule at any substitutable carbon ornitrogen atom. Unless otherwise specified, a bridged bicyclic group isoptionally substituted with one or more substituents as set forth foraliphatic groups. Additionally or alternatively, any substitutablenitrogen of a bridged bicyclic group is optionally substituted.Exemplary bridged bicyclics include:

The term “lower alkyl” refers to a C₁₋₄ straight or branched alkylgroup. Exemplary lower alkyl groups are methyl, ethyl, propyl,isopropyl, butyl, isobutyl, and tert-butyl.

The term “lower haloalkyl” refers to a C₁₋₄ straight or branched alkylgroup that is substituted with one or more halogen atoms.

The term “heteroatom” means one or more of oxygen, sulfur, nitrogen,phosphorus, or silicon (including, any oxidized form of nitrogen,sulfur, phosphorus, or silicon; the quaternized form of any basicnitrogen or; a substitutable nitrogen of a heterocyclic ring, forexample N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) orNR⁺ (as in N-substituted pyrrolidinyl)).

The term “unsaturated,” as used herein, means that a moiety has one ormore units of unsaturation.

As used herein, the term “bivalent C₁₋₈ (or C₁₋₆, C₂₋₈, C₂₋₆) saturatedor unsaturated, straight or branched, hydrocarbon chain”, refers tobivalent alkylene, alkenylene, and alkynylene chains that are straightor branched as defined herein.

The term “alkylene” refers to a bivalent alkyl group. An “alkylenechain” is a polymethylene group, i.e., —(CH₂)_(n)—, wherein n is apositive integer from 1 to 8, from 1 to 6, from 1 to 4, from 1 to 3,from 1 to 2, from 2 to 8, from 2 to 6, or from 2 to 3. A substitutedalkylene chain is a polymethylene group in which one or more methylenehydrogen atoms are replaced with a substituent. Suitable substituentsinclude those described below for a substituted aliphatic group.

The term “alkenylene” refers to a bivalent alkenyl group. A substitutedalkenylene chain is a polymethylene group containing at least one doublebond in which one or more hydrogen atoms are replaced with asubstituent. Suitable substituents include those described below for asubstituted aliphatic group.

As used herein, the term “cyclopropylenyl” refers to a bivalentcyclopropyl group of the following structure:

The term “halogen” means F, Cl, Br, or I.

The term “aryl” used alone or as part of a larger moiety as in“aralkyl,” “aralkoxy,” or “aryloxyalkyl,” refers to monocyclic orbicyclic ring systems having a total of five to fourteen ring members,wherein at least one ring in the system is aromatic and wherein eachring in the system contains 3 to 7 ring members. The term “aryl” may beused interchangeably with the term “aryl ring.”

The term “aryl” used alone or as part of a larger moiety as in“aralkyl,” “aralkoxy,” or “aryloxyalkyl,” refers to monocyclic andbicyclic ring systems having a total of five to 10 ring members, whereinat least one ring in the system is aromatic and wherein each ring in thesystem contains three to seven ring members. The term “aryl” may be usedinterchangeably with the term “aryl ring”. In certain embodiments of thepresent invention, “aryl” refers to an aromatic ring system whichincludes, but not limited to, phenyl, biphenyl, naphthyl, anthracyl andthe like, which may bear one or more substituents. Also included withinthe scope of the term “aryl,” as it is used herein, is a group in whichan aromatic ring is fused to one or more non-aromatic rings, such asindanyl, phthalimidyl, naphthimidyl, phenanthridinyl, ortetrahydronaphthyl, and the like.

The terms “heteroaryl” and “heteroar-,” used alone or as part of alarger moiety, e.g., “heteroaralkyl,” or “heteroaralkoxy,” refer togroups having 5 to 10 ring atoms, preferably 5, 6, or 9 ring atoms;having 6, 10, or 14π electrons shared in a cyclic array; and having, inaddition to carbon atoms, from one to five heteroatoms. The term“heteroatom” refers to nitrogen, oxygen, or sulfur, and includes anyoxidized form of nitrogen or sulfur, and any quaternized form of a basicnitrogen. Heteroaryl groups include, without limitation, thienyl,furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl,oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl,thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl,purinyl, naphthyridinyl, and pteridinyl. The terms “heteroaryl” and“heteroar-”, as used herein, also include groups in which aheteroaromatic ring is fused to one or more aryl, cycloaliphatic, orheterocyclyl rings, where the radical or point of attachment is on theheteroaromatic ring. Nonlimiting examples include indolyl, isoindolyl,benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl,benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl,quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl,phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl,tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one. Aheteroaryl group may be mono- or bicyclic. The term “heteroaryl” may beused interchangeably with the terms “heteroaryl ring,” “heteroarylgroup,” or “heteroaromatic,” any of which terms include rings that areoptionally substituted. The term “heteroaralkyl” refers to an alkylgroup substituted by a heteroaryl, wherein the alkyl and heteroarylportions independently are optionally substituted.

As used herein, the terms “heterocycle,” “heterocyclyl,” “heterocyclicradical,” and “heterocyclic ring” are used interchangeably and refer toa stable 5- to 7-membered monocyclic or 7-10-membered bicyclicheterocyclic moiety that is either saturated or partially unsaturated,and having, in addition to carbon atoms, one or more, preferably one tofour, heteroatoms, as defined above. When used in reference to a ringatom of a heterocycle, the term “nitrogen” includes a substitutednitrogen. As an example, in a saturated or partially unsaturated ringhaving 0-3 heteroatoms selected from oxygen, sulfur or nitrogen, thenitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as inpyrrolidinyl), or NR (as in N-substituted pyrrolidinyl).

A heterocyclic ring can be attached to its pendant group at anyheteroatom or carbon atom that results in a stable structure and any ofthe ring atoms can be optionally substituted. Examples of such saturatedor partially unsaturated heterocyclic radicals include, withoutlimitation, tetrahydrofuranyl, tetrahydrothiophenyl pyrrolidinyl,piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl,decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl,diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. Theterms “heterocycle,” “heterocyclyl,” “heterocyclyl ring,” “heterocyclicgroup,” “heterocyclic moiety,” and “heterocyclic radical,” are usedinterchangeably herein, and also include groups in which a heterocyclylring is fused to one or more aryl, heteroaryl, or cycloaliphatic rings,such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl, ortetrahydroquinolinyl, where the radical or point of attachment is on theheterocyclyl ring. A heterocyclyl group may be mono- or bicyclic. Theterm “heterocyclylalkyl” refers to an alkyl group substituted by aheterocyclyl, wherein the alkyl and heterocyclyl portions independentlyare optionally substituted.

As used herein, the term “partially unsaturated” refers to a ring moietythat includes at least one double or triple bond. The term “partiallyunsaturated” is intended to encompass rings having multiple sites ofunsaturation, but is not intended to include aryl or heteroarylmoieties, as herein defined.

As described herein, compounds of the invention may contain “optionallysubstituted” moieties. In general, the term “substituted,” whetherpreceded by the term “optionally” or not, means that one or morehydrogens of the designated moiety are replaced with a suitablesubstituent. Unless otherwise indicated, an “optionally substituted”group may have a suitable substituent at each substitutable position ofthe group, and when more than one position in any given structure may besubstituted with more than one substituent selected from a specifiedgroup, the substituent may be either the same or different at everyposition. Combinations of substituents envisioned by this invention arepreferably those that result in the formation of stable or chemicallyfeasible compounds. The term “stable,” as used herein, refers tocompounds that are not substantially altered when subjected toconditions to allow for their production, detection, and, in certainembodiments, their recovery, purification, and use for one or more ofthe purposes disclosed herein.

Suitable monovalent substituents on a substitutable carbon atom of an“optionally substituted” group are independently halogen;—(CH₂)₀₋₄R^(∘); —(CH₂)₀₋₄OR^(∘); —O(CH₂)₀₋₄R^(∘), —O—(CH₂)₀₋₄C(O)OR^(∘);—(CH₂)₀₋₄CH(OR^(∘))₂; —(CH₂)₀₋₄SR^(∘); —(CH₂)₀₋₄Ph, which may besubstituted with R^(∘); —(CH₂)₀₋₄O(CH₂)₀₋₁Ph which may be substitutedwith R^(∘); —CH═CHPh, which may be substituted with R^(∘);—(CH₂)₀₋₄O(CH₂)₀₋₁-pyridyl which may be substituted with R^(∘); —NO₂;—CN; —N₃; —(CH₂)₀₋₄N(R^(∘))₂; —(CH₂)₀₋₄N(R^(∘))C(O)R^(∘);—N(R^(∘))C(S)R^(∘); —(CH₂)₀₋₄N(R^(∘))C(O)NR^(∘) ₂; —N(R^(∘))C(S)NR^(∘)₂; —(CH₂)₀₋₄N(R^(∘))C(O)OR^(∘); —N(R^(∘))N(R^(∘))C(O)R^(∘);—N(R^(∘))N(R^(∘))C(O)NR^(∘) ₂; —N(R^(∘))N(R^(∘))C(O)OR^(∘);—(CH₂)₀₋₄C(O)R^(∘); —C(S)R^(∘); —(CH₂)₀₋₄C(O)OR^(∘);—(CH₂)₀₋₄C(O)SR^(∘); —(CH₂)₀₋₄C(O)OSiR^(∘) ₃; —(CH₂)₀₋₄OC(O)R^(∘);—OC(O)(CH₂)₀₋₄SR—, SC(S)SR^(∘); —(CH₂)₀₋₄SC(O)R^(∘); —(CH₂)₀₋₄C(O)NR^(∘)₂; —C(S)NR^(∘) ₂; —C(S)SR^(∘); —SC(S)SR^(∘), —(CH₂)₀₋₄OC(O)NR^(∘) ₂;—C(O)N(OR^(∘))R^(∘); —C(O)C(O)R^(∘); —C(O)CH₂C(O)R^(∘);—C(NOR^(∘))R^(∘); —(CH₂)₀₋₄SSR^(∘); —(CH₂)₀₋₄S(O)₂R^(∘);—(CH₂)₀₋₄S(O)₂OR^(∘); —(CH₂)₀₋₄OS(O)₂R^(∘); —S(O)₂NR^(∘) ₂,—(CH₂)₀₋₄S(O)R^(∘); —N(R^(∘))S(O)₂NR^(∘) ₂; —N(R^(∘))S(O)₂R^(∘);—N(OR^(∘))R^(∘); —C(NH)NR^(∘) ₂; —P(O)₂R^(∘); —P(O)R^(∘) ₂; —OP(O)R^(∘)₂; —OP(O)(OR^(∘))₂; SiR^(∘) ₃; —(C₁₋₄ straight or branchedalkylene)O—N(R^(∘))₂; or —(C₁₋₄ straight or branchedalkylene)C(O)O—N(R^(∘))₂, wherein each R^(∘) may be substituted asdefined below and is independently hydrogen, C₁₋₆ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, —CH₂-(5-6 membered heteroaryl ring), or a 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(∘), taken together with their intervening atom(s), form a3-12-membered saturated, partially unsaturated, or aryl mono- orbicyclic ring having 0-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, which may be substituted as defined below.

Suitable monovalent substituents on R^(∘) (or the ring formed by takingtwo independent occurrences of R^(∘) together with their interveningatoms), are independently halogen, —(CH₂)₀₋₂R^(●), -(haloR^(●)),—(CH₂)₀₋₂OH, —(CH₂)₀₋₂OR^(●), —(CH₂)₀₋₂CH(OR^(●))₂; —O(haloR^(●)), —CN,—N₃, —(CH₂)₀₋₂C(O)R^(●), —(CH₂)₀₋₂C(O)OH, —(CH₂)₀₋₂C(O)OR^(●),—(CH₂)₀₋₂SR^(●), —(CH₂)₀₋₂SH, —(CH₂)₀₋₂NH₂, —(CH₂)₀₋₂NHR^(●),—(CH₂)₀₋₂NR^(●) ₂, —NO₂, —SiR^(●) ₃, —OSiR^(●) ₃, —C(O)SR^(●), —(C₁₋₄straight or branched alkylene)C(O)OR^(●), or —SSR^(●) wherein each R^(●)is unsubstituted or where preceded by “halo” is substituted only withone or more halogens, and is independently selected from C₁₋₄ aliphatic,—CH₂Ph, —O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. Suitable divalent substituents on asaturated carbon atom of R^(∘) include ═O and ═S.

Suitable divalent substituents on a saturated carbon atom of an“optionally substituted” group include the following: ═O, ═S, ═NNR*₂,═NNHC(O)R*, ═NNHC(O)OR*, ═NNHS(O)₂R*, ═NR*, ═NOR*, —O(C(R*₂))₂₋₃O—, or—S(C(R*₂))₂₋₃S—, wherein each independent occurrence of R* is selectedfrom hydrogen, C₁₋₆ aliphatic which may be substituted as defined below,or an unsubstituted 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur. Suitable divalent substituents that are bound tovicinal substitutable carbons of an “optionally substituted” groupinclude: —O(CR*₂)₂₋₃O—, wherein each independent occurrence of R* isselected from hydrogen, C₁₋₆ aliphatic which may be substituted asdefined below, or an unsubstituted 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R* include halogen,—R^(●), -(haloR^(●)), —OH, —OR^(●), —O(haloR^(●)), —CN, —C(O)OH,—C(O)OR^(●), —NH₂, —NHR^(●), —NR^(●) ₂, or —NO₂, wherein each R^(●) isunsubstituted or where preceded by “halo” is substituted only with oneor more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

Suitable substituents on a substitutable nitrogen of an “optionallysubstituted” group include —R^(†), —NR^(†) ₂, —C(O)R^(†), —C(O)OR^(†),—C(O)C(O)R^(†), —C(O)CH₂C(O)R^(†), —S(O)₂R^(†), —S(O)₂NR^(†) ₂,—C(S)NR^(†) ₂, —C(NH)NR^(†) ₂, or —N(R^(†))S(O)₂R^(†); wherein eachR^(†) is independently hydrogen, C₁₋₆ aliphatic which may be substitutedas defined below, unsubstituted —OPh, or an unsubstituted 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(†), taken together with their intervening atom(s) form anunsubstituted 3-12-membered saturated, partially unsaturated, or arylmono- or bicyclic ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R^(†) are independentlyhalogen, —R^(●), -(haloR^(●)), —OH, —OR^(●), —O(haloR^(●)), —CN,—C(O)OH, —C(O)OR^(●), —NH₂, —NHR^(●), —NR^(●) ₂, or —NO₂, wherein eachR^(●) is unsubstituted or where preceded by “halo” is substituted onlywith one or more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example, S. M. Berge etal., describe pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein byreference. Pharmaceutically acceptable salts of the compounds of thisinvention include those derived from suitable inorganic and organicacids and bases. Examples of pharmaceutically acceptable, nontoxic acidaddition salts are salts of an amino group formed with inorganic acidssuch as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuricacid and perchloric acid or with organic acids such as acetic acid,oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid ormalonic acid or by using other methods used in the art such as ionexchange. Other pharmaceutically acceptable salts include adipate,alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptonate, glycerophosphate, gluconate,hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, pivalate,propionate, stearate, succinate, sulfate, tartrate, thiocyanate,p-toluenesulfonate, undecanoate, valerate salts, and the like.

Salts derived from appropriate bases include alkali metal, alkalineearth metal, ammonium and N⁺(C₁₋₄alkyl)₄ salts. Representative alkali oralkaline earth metal salts include sodium, lithium, potassium, calcium,magnesium, and the like. Further pharmaceutically acceptable saltsinclude, when appropriate, nontoxic ammonium, quaternary ammonium, andamine cations formed using counterions such as halide, hydroxide,carboxylate, sulfate, phosphate, nitrate, loweralkyl sulfonate and arylsulfonate.

Unless otherwise stated, structures depicted herein are also meant toinclude all isomeric (e.g., enantiomeric, diastereomeric, and geometric(or conformational)) forms of the structure; for example, the R and Sconfigurations for each asymmetric center, Z and E double bond isomers,and Z and E conformational isomers. Therefore, single stereochemicalisomers as well as enantiomeric, diastereomeric, and geometric (orconformational) mixtures of the present compounds are within the scopeof the invention. Unless otherwise stated, all tautomeric forms of thecompounds of the invention are within the scope of the invention.Additionally, unless otherwise stated, structures depicted herein arealso meant to include compounds that differ only in the presence of oneor more isotopically enriched atoms. For example, compounds having thepresent structures including the replacement of hydrogen by deuterium ortritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enriched carbonare within the scope of this invention. Such compounds are useful, forexample, as analytical tools, as probes in biological assays, or astherapeutic agents in accordance with the present invention. In certainembodiments, a warhead moiety, R³, of a provided compound comprises oneor more deuterium atoms.

As used herein, the term “irreversible” or “irreversible inhibitor”refers to an inhibitor (i.e. a compound) that is able to be covalentlybonded to HCV protease in a substantially non-reversible manner. Thatis, whereas a reversible inhibitor is able to bind to (but is generallyunable to form a covalent bond with) HCV protease, and therefore canbecome dissociated from the HCV protease an irreversible inhibitor willremain substantially bound to HCV protease once covalent bond formationhas occurred. Irreversible inhibitors usually display time dependency,whereby the degree of inhibition increases with the time with which theinhibitor is in contact with the enzyme. In certain embodiments, anirreversible inhibitor will remain substantially bound to HCV proteaseonce covalent bond formation has occurred and will remain bound for atime period that is longer than the life of the protein.

Methods for identifying if a compound is acting as an irreversibleinhibitor are known to one of ordinary skill in the art. Such methodsinclude, but are not limited to, enzyme kinetic analysis of theinhibition profile of the compound with HCV protease, the use of massspectrometry of the protein drug target modified in the presence of theinhibitor compound, discontinuous exposure, also known as “washout,”experiments, and the use of labeling, such as radiolabelled inhibitor,to show covalent modification of the enzyme, as well as other methodsknown to one of skill in the art.

One of ordinary skill in the art will recognize that certain reactivefunctional groups can act as “warheads.” As used herein, the term“warhead” or “warhead group” refers to a functional group present on acompound of the present invention wherein that functional group iscapable of covalently binding to an amino acid residue (such ascysteine, lysine, histidine, or other residues capable of beingcovalently modified) present in the binding pocket of the targetprotein, thereby irreversibly inhibiting the protein. It will beappreciated that the -L-Y group, as defined and described herein,provides such warhead groups for covalently, and irreversibly,inhibiting the protein.

As used herein, the term “inhibitor” is defined as a compound that bindsto and/or inhibits HCV protease with measurable affinity. In certainembodiments, an inhibitor has an IC₅₀ and/or binding constant of lessabout 50 μM, less than about 1 μM, less than about 500 nM, less thanabout 100 nM, less than about 10 nM, or less than about 1 nM.

A compound of the present invention may be tethered to a detectablemoiety. One of ordinary skill in the art will recognize that adetectable moiety may be attached to a provided compound via a suitablesubstituent. As used herein, the term “suitable substituent” refers to amoiety that is capable of covalent attachment to a detectable moiety.Such moieties are well known to one of ordinary skill in the art andinclude groups containing, e.g., a carboxylate moiety, an amino moiety,a thiol moiety, or a hydroxyl moiety, to name but a few. It will beappreciated that such moieties may be directly attached to a providedcompound or via a tethering group, such as a bivalent saturated orunsaturated hydrocarbon chain. In some embodiments, such moieties may beattached via click chemistry. In some embodiments, such moieties may beattached via a 1,3-cycloaddition of an azide with an alkyne, optionallyin the presence of a copper catalyst. Methods of using click chemistryare known in the art and include those described by Rostovtsev et al.,Angew. Chem. Int. Ed. 2002, 41, 2596-99 and Sun et al., BioconjugateChem., 2006, 17, 52-57.

As used herein, the term “detectable moiety” is used interchangeablywith the term “label” and relates to any moiety capable of beingdetected, e.g., primary labels and secondary labels. Primary labels,such as radioisotopes (e.g., tritium, ³²P, ³³P, 35S, or ¹⁴C), mass-tags,and fluorescent labels are signal generating reporter groups which canbe detected without further modifications. Detectable moieties alsoinclude luminescent and phosphorescent groups.

The term “secondary label” as used herein refers to moieties such asbiotin and various protein antigens that require the presence of asecond intermediate for production of a detectable signal. For biotin,the secondary intermediate may include streptavidin-enzyme conjugates.For antigen labels, secondary intermediates may include antibody-enzymeconjugates. Some fluorescent groups act as secondary labels because theytransfer energy to another group in the process of nonradiativefluorescent resonance energy transfer (FRET), and the second groupproduces the detected signal.

The terms “fluorescent label”, “fluorescent dye”, and “fluorophore” asused herein refer to moieties that absorb light energy at a definedexcitation wavelength and emit light energy at a different wavelength.Examples of fluorescent labels include, but are not limited to: AlexaFluor dyes (Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 532, AlexaFluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, AlexaFluor 660 and Alexa Fluor 680), AMCA, AMCA-S, BODIPY dyes (BODIPY FL,BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY 530/550, BODIPY 558/568,BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY650/665), Carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), Cascade Blue,Cascade Yellow, Coumarin 343, Cyanine dyes (Cy3, Cy5, Cy3.5, Cy5.5),Dansyl, Dapoxyl, Dialkylaminocoumarin,4′,5′-Dichloro-2′,7′-dimethoxy-fluorescein, DM-NERF, Eosin, Erythrosin,Fluorescein, FAM, Hydroxycoumarin, IRDyes (IRD40, IRD 700, IRD 800),JOE, Lissamine rhodamine B, Marina Blue, Methoxycoumarin,Naphthofluorescein, Oregon Green 488, Oregon Green 500, Oregon Green514, Pacific Blue, PyMPO, Pyrene, Rhodamine B, Rhodamine 6G, RhodamineGreen, Rhodamine Red, Rhodol Green,2′,4′,5′,7′-Tetra-bromosulfone-fluorescein, Tetramethyl-rhodamine (TMR),Carboxytetramethylrhodamine (TAMRA), Texas Red, Texas Red-X.

The term “mass-tag” as used herein refers to any moiety that is capableof being uniquely detected by virtue of its mass using mass spectrometry(MS) detection techniques. Examples of mass-tags include electrophorerelease tags such asN-[3-[4′-[(p-Methoxytetrafluorobenzyl)oxy]phenyl]-3-methylglyceronyl]isonipecoticAcid, 4′-[2,3,5,6-Tetrafluoro-4-(pentafluorophenoxyl)]methylacetophenone, and their derivatives. The synthesis and utility of thesemass-tags is described in U.S. Pat. Nos. 4,650,750, 4,709,016,5,360,8191, 5,516,931, 5,602,273, 5,604,104, 5,610,020, and 5,650,270.Other examples of mass-tags include, but are not limited to,nucleotides, dideoxynucleotides, oligonucleotides of varying length andbase composition, oligopeptides, oligosaccharides, and other syntheticpolymers of varying length and monomer composition. A large variety oforganic molecules, both neutral and charged (biomolecules or syntheticcompounds) of an appropriate mass range (100-2000 Daltons) may also beused as mass-tags.

The terms “measurable affinity” and “measurably inhibit,” as usedherein, means a measurable change in HCV protease activity between asample comprising a compound of the present invention, or compositionthereof, and HCV protease, and an equivalent sample comprising HCVprotease, in the absence of said compound, or composition thereof.

3. Description of Exemplary Compounds:

In certain embodiments, the present invention provides a compound offormula I:

-   or a pharmaceutically acceptable salt thereof, wherein:-   one of R^(a) and R^(b) is hydrogen and the other is —OH or —OC(O)R′,    or R^(a) and R^(b) are taken together to form an oxo group;-   R′ is an optionally substituted group selected from C₁₋₆ aliphatic,    C₃₋₇ cycloalkyl, 6-10 membered aryl, 5-10 membered heteroaryl having    1-4 heteroatoms independently selected from nitrogen, oxygen, or    sulfur, or 4-7 membered heterocyclyl having 1-2 heteroatoms    independently selected from nitrogen, oxygen, or sulfur;-   R^(x) and R^(y) are taken together to form an optionally substituted    C₃₋₇ membered ring having 0-2 heteroatoms independently selected    from nitrogen, oxygen, or sulfur;-   R¹ is an optionally substituted group selected from C₁₋₆ aliphatic    or C₃₋₇ cycloalkyl(C₁₋₃ alkyl);-   R² is hydrogen or an optionally substituted group selected from C₁₋₆    aliphatic or C₃₋₇ cycloalkyl;-   R³ is —(CH₂)_(n)-L-Y, wherein:    -   n is an integer from 0 to 5, inclusive;    -   L is a covalent bond or a bivalent C₁₋₈ saturated or        unsaturated, straight or branched, hydrocarbon chain, wherein        one, two, or three methylene units of L are optionally and        independently replaced by cyclopropylene, —NR—, —N(R)C(O)—,        —C(O)N(R)—, —N(R)SO₂—, —SO₂N(R)—, —O—, —C(O)—, —OC(O)—, —C(O)O—,        —S—, —SO—, —SO₂—, —C(═S)—, —C(═NR)—, —N═N—, or —C(═N₂)—;    -   Y is hydrogen, C₁₋₆ aliphatic optionally substituted with oxo,        halogen, NO₂, or CN, or a 3-10 membered monocyclic or bicyclic,        saturated, partially unsaturated, or aryl ring having 0-3        heteroatoms independently selected from nitrogen, oxygen, or        sulfur, and wherein said ring is substituted with 1-4 R^(e)        groups; and    -   each R^(e) is independently selected from -Q-Z, oxo, NO₂,        halogen, CN, a suitable leaving group, or C₁₋₆ aliphatic        optionally substituted with oxo, halogen, NO₂, or CN, wherein:        -   Q is a covalent bond or a bivalent C₁₋₆ saturated or            unsaturated, straight or branched, hydrocarbon chain,            wherein one or two methylene units of Q are optionally and            independently replaced by —N(R)—, —S—, —O—, —C(O)—, —OC(O)—,            —C(O)O—, —SO—, or —SO₂—, —N(R)C(O)—, —C(O)N(R)—, —N(R)SO₂—,            or —SO₂N(R)—; and        -   Z is hydrogen or C₁₋₆ aliphatic optionally substituted with            oxo, halogen, NO₂, or CN;-   R⁴ is —NHC(O)NHR⁵, —NHC(O)OR⁶, or

-   R⁵ is an optionally substituted group selected from C₁₋₆ aliphatic,    bridged bicyclic, 6-10 membered aryl, 5-10 membered heteroaryl    having 1-4 heteroatoms independently selected from nitrogen, oxygen,    or sulfur, or 4-7 membered heterocyclyl having 1-2 heteroatoms    independently selected from nitrogen, oxygen, or sulfur;-   R⁶ is an optionally substituted group selected from C₁₋₆ aliphatic,    bridged bicyclic, 6-10 membered aryl, 5-10 membered heteroaryl    having 1-4 heteroatoms independently selected from nitrogen, oxygen,    or sulfur, or 4-7 membered heterocyclyl having 1-2 heteroatoms    independently selected from nitrogen, oxygen, or sulfur;-   R⁷ is an optionally substituted group selected from C₁₋₆ aliphatic,    bridged bicyclic, 6-10 membered aryl, 5-10 membered heteroaryl    having 1-4 heteroatoms independently selected from nitrogen, oxygen,    or sulfur, or 4-7 membered heterocyclyl having 1-2 heteroatoms    independently selected from nitrogen, oxygen, or sulfur; and-   R is hydrogen or an optionally substituted group selected from C₁₋₆    aliphatic, C₃₋₇ cycloalkyl, 6-10 membered aryl, 5-10 membered    heteroaryl having 1-4 heteroatoms independently selected from    nitrogen, oxygen, or sulfur, or 4-7 membered heterocyclyl having 1-2    heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In certain embodiments, n is an integer from 1 to 5, inclusive. Incertain embodiments, n is 0. In certain embodiments, n is 1. In certainembodiments, n is 2. In certain embodiments, n is 3. In certainembodiments, n is 4. In certain embodiments, n is 5.

In certain embodiments, L is a covalent bond.

In certain embodiments, L is a bivalent C₁₋₈ saturated or unsaturated,straight or branched, hydrocarbon chain. In certain embodiments, L is—CH₂—.

In certain embodiments, L is a covalent bond, —CH₂—, —NH—, —CH₂NH—,—NHCH₂—, —NHC(O)—, —NHC(O)CH₂OC(O)—, —CH₂NHC(O)—, —NHSO₂—, —NHSO₂CH₂—,—NHC(O)CH₂OC(O)—, or —SO₂NH—.

In some embodiments, L is a bivalent C₂₋₈ straight or branched,hydrocarbon chain wherein L has at least one double bond and one or twoadditional methylene units of L are optionally and independentlyreplaced by —NRC(O)—, —C(O)NR—, —N(R)SO₂—, —SO₂N(R)—, —S—, —S(O)—,—SO₂—, —OC(O)—, —C(O)O—, cyclopropylene, —O—, —N(R)—, or —C(O)—.

In certain embodiments, L is a bivalent C₂₋₈ straight or branched,hydrocarbon chain wherein L has at least one double bond and at leastone methylene unit of L is replaced by —C(O)—, —NRC(O)—, —C(O)NR—,—N(R)SO₂—, —SO₂N(R)—, —S—, —S(O)—, —SO₂—, —OC(O)—, or —C(O)O—, and oneor two additional methylene units of L are optionally and independentlyreplaced by cyclopropylene, —O—, —N(R)—, or —C(O)—.

In some embodiments, L is a bivalent C₂₋₈ straight or branched,hydrocarbon chain wherein L has at least one double bond and at leastone methylene unit of L is replaced by —C(O)—, and one or two additionalmethylene units of L are optionally and independently replaced bycyclopropylene, —O—, —N(R)—, or —C(O)—.

As described above, in certain embodiments, L is a bivalent C₂₋₈straight or branched, hydrocarbon chain wherein L has at least onedouble bond. One of ordinary skill in the art will recognize that such adouble bond may exist within the hydrocarbon chain backbone or may be“exo” to the backbone chain and thus forming an alkylidene group. By wayof example, such an L group having an alkylidene branched chain includes—CH₂C(═CH₂)CH₂—. Thus, in some embodiments, L is a bivalent C₂₋₈straight or branched, hydrocarbon chain wherein L has at least onealkylidenyl double bond. Exemplary L groups include —NHC(O)C(═CH₂)CH₂—.

In certain embodiments, L is a bivalent C₂₋₈ straight or branched,hydrocarbon chain wherein L has at least one double bond and at leastone methylene unit of L is replaced by —C(O)—. In certain embodiments, Lis —C(O)CH═CH(CH₃)—, —C(O)CH═CHCH₂NH(CH₃)—, —C(O)CH═CH(CH₃)—,—C(O)CH═CH—, —CH₂C(O)CH═CH—, —CH₂C(O)CH═CH(CH₃)—, —CH₂CH₂C(O)CH═CH—,—CH₂CH₂C(O)CH═CHCH₂—, —CH₂CH₂C(O)CH═CHCH₂NH(CH₃)—, or—CH₂CH₂C(O)CH═CH(CH₃)—, or —CH(CH₃)OC(O)CH═CH—.

In certain embodiments, L is a bivalent C₂₋₈ straight or branched,hydrocarbon chain wherein L has at least one double bond and at leastone methylene unit of L is replaced by —OC(O)—.

In some embodiments, L is a bivalent C₂₋₈ straight or branched,hydrocarbon chain wherein L has at least one double bond and at leastone methylene unit of L is replaced by —NRC(O)—, —C(O)NR—, —N(R)SO₂—,—SO₂N(R)—, —S—, —S(O)—, —SO₂—, —OC(O)—, or —C(O)O—, and one or twoadditional methylene units of L are optionally and independentlyreplaced by cyclopropylene, —O—, —N(R)—, or —C(O)—. In some embodiments,L is —CH₂OC(O)CH═CHCH₂—, —CH₂—OC(O)CH═CH—, or —CH(CH═CH₂)OC(O)CH═CH—.

In certain embodiments, L is —NRC(O)CH═CH—, —NRC(O)CH═CHCH₂N(CH₃)—,—NRC(O)CH═CHCH₂O—, —CH₂NRC(O)CH═CH—, —NRSO₂CH═CH—, —NRSO₂CH═CHCH₂—,—NRC(O)(C═N₂)C(O)—, —NRC(O)CH═CHCH₂N(CH₃)—, —NRSO₂CH═CH—,—NRSO₂CH═CHCH₂—, —NRC(O)CH═CHCH₂O—, —NRC(O)C(═CH₂)CH₂—, —CH₂NRC(O)—,—CH₂NRC(O)CH═CH—, —CH₂CH₂NRC(O)—, or —CH₂NRC(O)cyclopropylene-, whereineach R is independently hydrogen or optionally substituted C₁₋₆aliphatic.

In certain embodiments, L is —NHC(O)CH═CH—, —NHC(O)CH═CHCH₂N(CH₃)—,—NHC(O)CH═CHCH₂O—, —CH₂NHC(O)CH═CH—, —NHSO₂CH═CH—, —NHSO₂CH═CHCH₂—,—NHC(O)(C═N₂)C(O)—, —NHC(O)CH═CHCH₂N(CH₃)—, —NHSO₂CH═CH—,—NHSO₂CH═CHCH₂—, —NHC(O)CH═CHCH₂O—, —NHC(O)C(═CH₂)CH₂—, —CH₂NHC(O)—,—CH₂NHC(O)CH═CH—, —CH₂CH₂NHC(O)—, or —CH₂NHC(O)cyclopropylene-.

In some embodiments, L is a bivalent C₂₋₈ straight or branched,hydrocarbon chain wherein L has at least one triple bond. In certainembodiments, L is a bivalent C₂₋₈ straight or branched, hydrocarbonchain wherein L has at least one triple bond and one or two additionalmethylene units of L are optionally and independently replaced by—NRC(O)—, —C(O)NR—, —S—, —S(O)—, —SO₂—, —C(═S)—, —C(═NR)—, —O—, —N(R)—,or —C(O)—. In some embodiments, L has at least one triple bond and atleast one methylene unit of L is replaced by —N(R)—, —N(R)C(O)—, —C(O)—,—C(O)O—, or —OC(O)—, or —O—.

Exemplary L groups include —C≡C—, —C≡CCH₂N(isopropyl)-,—NHC(O)C≡CCH₂CH₂—, —CH₂—C≡C—CH₂—, —C≡CCH₂O—, —CH₂C(O)C≡C—, —C(O)C≡C—, or—CH₂OC(═O)C≡C—.

In certain embodiments, L is a bivalent C₂₋₈ straight or branched,hydrocarbon chain wherein one methylene unit of L is replaced bycyclopropylene and one or two additional methylene units of L areindependently replaced by —C(O)—, —NRC(O)—, —C(O)NR—, —N(R)SO₂—, or—SO₂N(R)—. Exemplary L groups include —NHC(O)-cyclopropylene-SO₂— and—NHC(O)-cyclopropylene-.

As defined generally above, Y is hydrogen, C₁₋₆ aliphatic optionallysubstituted with oxo, halogen, NO₂, or CN, or a 3-10 membered monocyclicor bicyclic, saturated, partially unsaturated, or aryl ring having 0-3heteroatoms independently selected from nitrogen, oxygen, or sulfur, andwherein said ring is substituted with at 1-4 R^(e) groups, each R^(e) isindependently selected from -Q-Z, oxo, NO₂, halogen, CN, or C₁₋₆aliphatic, wherein Q is a covalent bond or a bivalent C₁₋₆ saturated orunsaturated, straight or branched, hydrocarbon chain, wherein one or twomethylene units of Q are optionally and independently replaced by—N(R)—, —S—, —O—, —C(O)—, —OC(O)—, —C(O)O—, —SO—, or —SO₂—, —N(R)C(O)—,—C(O)N(R)—, —N(R)SO₂—, or —SO₂N(R)—; and, Z is hydrogen or C₁₋₆aliphatic optionally substituted with oxo, halogen, NO₂, or CN.

In certain embodiments, Y is hydrogen.

In certain embodiments, Y is C₁₋₆ aliphatic optionally substituted withoxo, halogen, NO₂, or CN. In some embodiments, Y is C₂₋₆ alkenyloptionally substituted with oxo, halogen, NO₂, or CN. In otherembodiments, Y is C₂₋₆ alkynyl optionally substituted with oxo, halogen,NO₂, or CN. In some embodiments, Y is C₂₋₆ alkenyl. In otherembodiments, Y is C₂₋₄ alkynyl.

In other embodiments, Y is C₁₋₆ alkyl substituted with oxo, halogen,NO₂, or CN. Such Y groups include —CH₂F, —CH₂Cl, —CH₂CN, and —CH₂NO₂.

In certain embodiments, Y is a saturated 3-6 membered monocyclic ringhaving 0-3 heteroatoms independently selected from nitrogen, oxygen, orsulfur, wherein Y is substituted with 1-4 R^(e) groups, wherein eachR^(e) is as defined above and described herein.

In some embodiments, Y is a saturated 3-4 membered heterocyclic ringhaving 1 heteroatom selected from oxygen or nitrogen wherein said ringis substituted with 1-2 R^(e) groups, wherein each R^(e) is as definedabove and described herein. Exemplary such rings are epoxide and oxetanerings, wherein each ring is substituted with 1-2 R^(e) groups, whereineach R^(e) is as defined above and described herein.

In other embodiments, Y is a saturated 5-6 membered heterocyclic ringhaving 1-2 heteroatom selected from oxygen or nitrogen wherein said ringis substituted with 1-4 R^(e) groups, wherein each R^(e) is as definedabove and described herein. Such rings include piperidine andpyrrolidine, wherein each ring is substituted with 1-4 R^(e) groups,wherein each R^(e) is as defined above and described herein. In certainembodiments, Y is

wherein each R, Q, Z, and R^(e) is as defined above and describedherein.

In some embodiments, Y is a saturated 3-6 membered carbocyclic ring,wherein said ring is substituted with 1-4 R^(e) groups, wherein eachR^(e) is as defined above and described herein. In certain embodiments,Y is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, wherein eachring is substituted with 1-4 R^(e) groups, wherein each R^(e) is asdefined above and described herein. In certain embodiments, Y is

wherein R^(e) is as defined above and described herein. In certainembodiments, Y is cyclopropyl optionally substituted with halogen, CN orNO₂.

In certain embodiments, Y is a partially unsaturated 3-6 memberedmonocyclic ring having 0-3 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, wherein said ring is substituted with 1-4R^(e) groups, wherein each R^(e) is as defined above and describedherein.

In some embodiments, Y is a partially unsaturated 3-6 memberedcarbocyclic ring, wherein said ring is substituted with 1-4 R^(e)groups, wherein each R^(e) is as defined above and described herein. Insome embodiments, Y is cyclopropenyl, cyclobutenyl, cyclopentenyl, orcyclohexenyl wherein each ring is substituted with 1-4 R^(e) groups,wherein each R^(e) is as defined above and described herein. In certainembodiments, Y is

wherein each R^(e) is as defined above and described herein.

In certain embodiments, Y is a partially unsaturated 4-6 memberedheterocyclic ring having 1-2 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, wherein said ring is substituted with 1-4R^(e) groups, wherein each R^(e) is as defined above and describedherein. In certain embodiments, Y is selected from:

wherein each R and R^(e) is as defined above and described herein.

In certain embodiments, Y is a 6-membered aromatic ring having 0-2nitrogens wherein said ring is substituted with 1-4 R^(e) groups,wherein each R^(e) group is as defined above and described herein. Incertain embodiments, Y is phenyl, pyridyl, or pyrimidinyl, wherein eachring is substituted with 1-4 R^(e) groups, wherein each R^(e) is asdefined above and described herein.

In some embodiments, Y is selected from:

wherein each R^(e) is as defined above and described herein.

In other embodiments, Y is a 5-membered heteroaryl ring having 1-3heteroatoms independently selected from nitrogen, oxygen, or sulfur,wherein said ring is substituted with 1-3 R^(e) groups, wherein eachR^(e) group is as defined above and described herein. In someembodiments, Y is a 5 membered partially unsaturated or aryl ring having1-3 heteroatoms independently selected from nitrogen, oxygen, andsulfur, wherein said ring is substituted with 1-4 R^(e) groups, whereineach R^(e) group is as defined above and described herein. Exemplarysuch rings are isoxazolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl,pyrrolyl, furanyl, thienyl, triazole, thiadiazole, and oxadiazole,wherein each ring is substituted with 1-3 R^(e) groups, wherein eachR^(e) group is as defined above and described herein. In certainembodiments, Y is selected from:

wherein each R and R^(e) is as defined above and described herein.

In certain embodiments, Y is an 8-10 membered bicyclic, saturated,partially unsaturated, or aryl ring having 0-3 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur, wherein said ring issubstituted with 1-4 R^(e) groups, wherein R^(e) is as defined above anddescribed herein. According to another aspect, Y is a 9-10 memberedbicyclic, partially unsaturated, or aryl ring having 1-3 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, wherein saidring is substituted with 1-4 R^(e) groups, wherein R^(e) is as definedabove and described herein. Exemplary such bicyclic rings include2,3-dihydrobenzo[d]isothiazole, wherein said ring is substituted with1-4 R^(e) groups, wherein R^(e) is as defined above and describedherein.

As defined generally above, each R^(e) group is independently selectedfrom -Q-Z, oxo, NO₂, halogen, CN, a suitable leaving group, or C₁₋₆aliphatic optionally substituted with oxo, halogen, NO₂, or CN, whereinQ is a covalent bond or a bivalent C₁₋₆ saturated or unsaturated,straight or branched, hydrocarbon chain, wherein one or two methyleneunits of Q are optionally and independently replaced by —N(R)—, —S—,—O—, —C(O)—, —OC(O)—, —C(O)O—, —SO—, or —SO₂—, —N(R)C(O)—, —C(O)N(R)—,—N(R)SO₂—, or —SO₂N(R)—; and Z is hydrogen or C₁₋₆ aliphatic optionallysubstituted with oxo, halogen, NO₂, or CN.

In certain embodiments, R^(e) is C₁₋₆ aliphatic optionally substitutedwith oxo, halogen, NO₂, or CN. In other embodiments, R^(e) is oxo, NO₂,halogen, or CN.

In some embodiments, R^(e) is -Q-Z, wherein Q is a covalent bond and Zis hydrogen (i.e., R^(e) is hydrogen). In other embodiments, R^(e) is-Q-Z, wherein Q is a bivalent C₁₋₆ saturated or unsaturated, straight orbranched, hydrocarbon chain, wherein one or two methylene units of Q areoptionally and independently replaced by —NR—, —NRC(O)—, —C(O)NR—, —S—,—O—, —C(O)—, —SO—, or —SO₂—. In other embodiments, Q is a bivalent C₂₋₆straight or branched, hydrocarbon chain having at least one double bond,wherein one or two methylene units of Q are optionally and independentlyreplaced by —NR—, —NRC(O)—, —C(O)NR—, —S—, —O—, —C(O)—, —SO—, or —SO₂—.In certain embodiments, the Z moiety of the R^(e) group is hydrogen. Insome embodiments, -Q-Z is —NHC(O)CH═CH₂ or —C(O)CH═CH₂.

In certain embodiments, each R^(e) is independently selected from fromoxo, NO₂, CN, fluoro, chloro, —NHC(O)CH═CH₂, —C(O)CH═CH₂, —CH₂CH═CH₂,—C≡CH, —C(O)OCH₂Cl, —C(O)OCH₂F, —C(O)OCH₂CN, —C(O)CH₂Cl, —C(O)CH₂F,—C(O)CH₂CN, or —CH₂C(O)CH₃.

In certain embodiments, R^(e) is a suitable leaving group, ie a groupthat is subject to nucleophilic displacement. A “suitable leaving” is achemical group that is readily displaced by a desired incoming chemicalmoiety such as the thiol moiety of a cysteine of interest. Suitableleaving groups are well known in the art, e.g., see, “Advanced OrganicChemistry,” Jerry March, 5th Ed., pp. 351-357, John Wiley and Sons, N.Y.Such leaving groups include, but are not limited to, halogen, alkoxy,sulphonyloxy, optionally substituted alkylsulphonyloxy, optionallysubstituted alkenylsulfonyloxy, optionally substituted arylsulfonyloxy,acyl, and diazonium moieties. Examples of suitable leaving groupsinclude chloro, iodo, bromo, fluoro, acetyl,methanesulfonyloxy(mesyloxy), tosyloxy, triflyloxy,nitro-phenylsulfonyloxy(nosyloxy), andbromo-phenylsulfonyloxy(brosyloxy).

In certain embodiments, the following embodiments and combinations of-L-Y apply:

-   -   (a) L is a bivalent C₂₋₈ straight or branched, hydrocarbon chain        wherein L has at least one double bond and one or two additional        methylene units of L are optionally and independently replaced        by —NRC(O)—, —C(O)NR—, —N(R)SO₂—, —SO₂N(R)—, —S—, —S(O)—, —SO₂—,        —OC(O)—, —C(O)O—, cyclopropylene, —O—, —N(R)—, or —C(O)—; and Y        is hydrogen or C₁₋₆ aliphatic optionally substituted with oxo,        halogen, NO₂, or CN; or    -   (b) L is a bivalent C₂₋₈ straight or branched, hydrocarbon chain        wherein L has at least one double bond and at least one        methylene unit of L is replaced by —C(O)—, —NRC(O)—, —C(O)NR—,        —N(R)SO₂—, —SO₂N(R)—, —S—, —S(O)—, —SO₂—, —OC(O)—, or —C(O)O—,        and one or two additional methylene units of L are optionally        and independently replaced by cyclopropylene, —O—, —N(R)—, or        —C(O)—; and Y is hydrogen or C₁₋₆ aliphatic optionally        substituted with oxo, halogen, NO₂, or CN; or    -   (c) L is a bivalent C₂₋₈ straight or branched, hydrocarbon chain        wherein L has at least one double bond and at least one        methylene unit of L is replaced by —C(O)—, and one or two        additional methylene units of L are optionally and independently        replaced by cyclopropylene, —O—, —N(R)—, or —C(O)—; and Y is        hydrogen or C₁₋₆ aliphatic optionally substituted with oxo,        halogen, NO₂, or CN; or    -   (d) L is a bivalent C₂₋₈ straight or branched, hydrocarbon chain        wherein L has at least one double bond and at least one        methylene unit of L is replaced by —C(O)—; and Y is hydrogen or        C₁₋₆ aliphatic optionally substituted with oxo, halogen, NO₂, or        CN; or    -   (e) L is a bivalent C₂₋₈ straight or branched, hydrocarbon chain        wherein L has at least one double bond and at least one        methylene unit of L is replaced by —OC(O)—; and Y is hydrogen or        C₁₋₆ aliphatic optionally substituted with oxo, halogen, NO₂, or        CN; or    -   (f) L is —NRC(O)CH═CH—, —NRC(O)CH═CHCH₂N(CH₃)—,        —NRC(O)CH═CHCH₂O—, —CH₂NRC(O)CH═CH—, —NRSO₂CH═CH—,        —NRSO₂CH═CHCH₂—, —NRC(O)(C═N₂)—, —NRC(O)(C═N₂)C(O)—,        —NRC(O)CH═CHCH₂N(CH₃)—, —NRSO₂CH═CH—, —NRSO₂CH═CHCH₂—,        —NRC(O)CH═CHCH₂O—, —NRC(O)C(═CH₂)CH₂—, —CH₂NRC(O)—,        —CH₂NRC(O)CH═CH—, —CH₂CH₂NRC(O)—, or —CH₂NRC(O)cyclopropylene-;        wherein R is H or optionally substituted C₁₋₆ aliphatic; and Y        is hydrogen or C₁₋₆ aliphatic optionally substituted with oxo,        halogen, NO₂, or CN; or    -   (g) L is —NHC(O)CH═CH—, —NHC(O)CH═CHCH₂N(CH₃)—,        —NHC(O)CH═CHCH₂O—, —CH₂NHC(O)CH═CH—, —NHSO₂CH═CH—,        —NHSO₂CH═CHCH₂—, —NHC(O)(C═N₂)—, —NHC(O)(C═N₂)C(O)—,        —NHC(O)CH═CHCH₂N(CH₃)—, —NHSO₂CH═CH—, —NHSO₂CH═CHCH₂—,        —NHC(O)CH═CHCH₂O—, —NHC(O)C(=CH₂)CH₂—, —CH₂NHC(O)—,        —CH₂NHC(O)CH═CH—, —CH₂CH₂NHC(O)—, or —CH₂NHC(O)cyclopropylene-;        and Y is hydrogen or C₁₋₆ aliphatic optionally substituted with        oxo, halogen, NO₂, or CN; or    -   (h) L is a bivalent C₂₋₈ straight or branched, hydrocarbon chain        wherein L has at least one alkylidenyl double bond and at least        one methylene unit of L is replaced by —C(O)—, —NRC(O)—,        —C(O)NR—, —N(R)SO₂—, —SO₂N(R)—, —S—, —S(O)—, —SO₂—, —OC(O)—, or        —C(O)O—, and one or two additional methylene units of L are        optionally and independently replaced by cyclopropylene, —O—,        —N(R)—, or —C(O)—; and Y is hydrogen or C₁₋₆ aliphatic        optionally substituted with oxo, halogen, NO₂, or CN; or    -   (i) L is a bivalent C₂₋₈ straight or branched, hydrocarbon chain        wherein L has at least one triple bond and one or two additional        methylene units of L are optionally and independently replaced        by —NRC(O)—, —C(O)NR—, —N(R)SO₂—, —SO₂N(R)—, —S—, —S(O)—, —SO₂—,        —OC(O)—, or —C(O)O—, and Y is hydrogen or C₁₋₆ aliphatic        optionally substituted with oxo, halogen, NO₂, or CN; or    -   (j) L is —C≡C—, —C≡CCH₂N(isopropyl)-, —NHC(O)C≡CCH₂CH₂—,        —CH₂—C≡C—CH₂—, —C≡CCH₂O—, —CH₂C(O)C≡C—, —C(O)C≡C—, or        —CH₂OC(═O)C≡C—; and Y is hydrogen or C₁₋₆ aliphatic optionally        substituted with oxo, halogen, NO₂, or CN; or    -   (k) L is a bivalent C₂₋₈ straight or branched, hydrocarbon chain        wherein one methylene unit of L is replaced by cyclopropylene        and one or two additional methylene units of L are independently        replaced by —NRC(O)—, —C(O)NR—, —N(R)SO₂—, —SO₂N(R)—, —S—,        —S(O)—, —SO₂—, —OC(O)—, or —C(O)O—; and Y is hydrogen or C₁₋₆        aliphatic optionally substituted with oxo, halogen, NO₂, or CN;        or    -   (l) L is a covalent bond and Y is selected from:        -   (i) C₁₋₆ alkyl substituted with oxo, halogen, NO₂, or CN;        -   (ii) C₂₋₆ alkenyl optionally substituted with oxo, halogen,            NO₂, or CN; or        -   (iii) C₂₋₆ alkynyl optionally substituted with oxo, halogen,            NO₂, or CN; or        -   (iv) a saturated 3-4 membered heterocyclic ring having 1            heteroatom selected from oxygen or nitrogen wherein said            ring is substituted with 1-2 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or    -   (v) a saturated 5-6 membered heterocyclic ring having 1-2        heteroatom selected from oxygen or nitrogen wherein said ring is        substituted with 1-4 R^(e) groups, wherein each R^(e) is as        defined above and described herein; or        -   (vi)

wherein each R, Q, Z, and R^(e) is as defined above and describedherein; or

-   -   -   (vii) a saturated 3-6 membered carbocyclic ring, wherein            said ring is substituted with 1-4 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (viii) a partially unsaturated 3-6 membered monocyclic ring            having 0-3 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, wherein said ring is substituted with 1-4            R^(e) groups, wherein each R^(e) is as defined above and            described herein; or        -   (ix) a partially unsaturated 3-6 membered carbocyclic ring,            wherein said ring is substituted with 1-4 R^(e) groups,            wherein each R^(e) is as defined above and described herein;            or        -   (x)

wherein each R^(e) is as defined above and described herein; or

-   -   -   (xi) a partially unsaturated 4-6 membered heterocyclic ring            having 1-2 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, wherein said ring is substituted with 1-4            R^(e) groups, wherein each R^(e) is as defined above and            described herein; or        -   (xii)

wherein each R and R^(e) is as defined above and described herein; or

-   -   -   (xiii) a 6-membered aromatic ring having 0-2 nitrogens            wherein said ring is substituted with 1-4 R^(e) groups,            wherein each R^(e) group is as defined above and described            herein; or        -   (xiv)

wherein each R^(e) is as defined above and described herein; or

-   -   -   (xv) a 5-membered heteroaryl ring having 1-3 heteroatoms            independently selected from nitrogen, oxygen, or sulfur,            wherein said ring is substituted with 1-3 R^(e) groups,            wherein each R^(e) group is as defined above and described            herein; or        -   (xvi)

wherein each R and R^(e) is as defined above and described herein; or

-   -   -   (xvii) an 8-10 membered bicyclic, saturated, partially            unsaturated, or aryl ring having 0-3 heteroatoms            independently selected from nitrogen, oxygen, or sulfur,            wherein said ring is substituted with 1-4 R^(e) groups,            wherein R^(e) is as defined above and described herein;

    -   (m) L is —C(O)— and Y is selected from:        -   (i) C₁₋₆ alkyl substituted with oxo, halogen, NO₂, or CN; or        -   (ii) C₂₋₆ alkenyl optionally substituted with oxo, halogen,            NO₂, or CN; or        -   (iii) C₂₋₆ alkynyl optionally substituted with oxo, halogen,            NO₂, or CN; or        -   (iv) a saturated 3-4 membered heterocyclic ring having 1            heteroatom selected from oxygen or nitrogen wherein said            ring is substituted with 1-2 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (v) a saturated 5-6 membered heterocyclic ring having 1-2            heteroatom selected from oxygen or nitrogen wherein said            ring is substituted with 1-4 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (vi)

wherein each R, Q, Z, and R^(e) is as defined above and describedherein; or

-   -   -   (vii) a saturated 3-6 membered carbocyclic ring, wherein            said ring is substituted with 1-4 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (viii) a partially unsaturated 3-6 membered monocyclic ring            having 0-3 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, wherein said ring is substituted with 1-4            R^(e) groups, wherein each R^(e) is as defined above and            described herein; or        -   (ix) a partially unsaturated 3-6 membered carbocyclic ring,            wherein said ring is substituted with 1-4 R^(e) groups,            wherein each R^(e) is as defined above and described herein:        -   (x)

wherein each R^(e) is as defined above and described herein; or

-   -   -   (xi) a partially unsaturated 4-6 membered heterocyclic ring            having 1-2 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, wherein said ring is substituted with 1-4            R^(e) groups, wherein each R^(e) is as defined above and            described herein; or        -   (xii)

wherein each R and R^(e) is as defined above and described herein; or

-   -   -   (xiii) a 6-membered aromatic ring having 0-2 nitrogens            wherein said ring is substituted with 1-4 R^(e) groups,            wherein each R^(e) group is as defined above and described            herein; or        -   (xiv)

wherein each R^(e) is as defined above and described herein; or

-   -   -   (xv) a 5-membered heteroaryl ring having 1-3 heteroatoms            independently selected from nitrogen, oxygen, or sulfur,            wherein said ring is substituted with 1-3 R^(e) groups,            wherein each R^(e) group is as defined above and described            herein; or        -   (xvi)

wherein each R and R^(e) is as defined above and described herein; or

-   -   -   (xvii) an 8-10 membered bicyclic, saturated, partially            unsaturated, or aryl ring having 0-3 heteroatoms            independently selected from nitrogen, oxygen, or sulfur,            wherein said ring is substituted with 1-4 R^(e) groups,            wherein R^(e) is as defined above and described herein;

    -   (n) L is —N(R)C(O)— and Y is selected from:        -   (i) C₁₋₆ alkyl substituted with oxo, halogen, NO₂, or CN; or        -   (ii) C₂₋₆ alkenyl optionally substituted with oxo, halogen,            NO₂, or CN; or        -   (iii) C₂₋₆ alkynyl optionally substituted with oxo, halogen,            NO₂, or CN; or        -   (iv) a saturated 3-4 membered heterocyclic ring having 1            heteroatom selected from oxygen or nitrogen wherein said            ring is substituted with 1-2 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (v) a saturated 5-6 membered heterocyclic ring having 1-2            heteroatom selected from oxygen or nitrogen wherein said            ring is substituted with 1-4 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (vi)

wherein each R, Q, Z, and R^(e) is as defined above and describedherein; or

-   -   -   (vii) a saturated 3-6 membered carbocyclic ring, wherein            said ring is substituted with 1-4 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (viii) a partially unsaturated 3-6 membered monocyclic ring            having 0-3 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, wherein said ring is substituted with 1-4            R^(e) groups, wherein each R^(e) is as defined above and            described herein; or        -   (ix) a partially unsaturated 3-6 membered carbocyclic ring,            wherein said ring is substituted with 1-4 R^(e) groups,            wherein each R^(e) is as defined above and described herein;        -   (x)

wherein each R^(e) is as defined above and described herein; or

-   -   -   (xi) a partially unsaturated 4-6 membered heterocyclic ring            having 1-2 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, wherein said ring is substituted with 1-4            R^(e) groups, wherein each R^(e) is as defined above and            described herein; or        -   (xii)

wherein each R and R^(e) is as defined above and described herein; or

-   -   -   (xiii) a 6-membered aromatic ring having 0-2 nitrogens            wherein said ring is substituted with 1-4 R^(e) groups,            wherein each R^(e) group is as defined above and described            herein; or        -   (xiv)

wherein each R^(e) is as defined above and described herein; or

-   -   -   (xv) a 5-membered heteroaryl ring having 1-3 heteroatoms            independently selected from nitrogen, oxygen, or sulfur,            wherein said ring is substituted with 1-3 R^(e) groups,            wherein each R^(e) group is as defined above and described            herein; or        -   (xvi)

wherein each R and R^(e) is as defined above and described herein; or

-   -   -   (xvii) an 8-10 membered bicyclic, saturated, partially            unsaturated, or aryl ring having 0-3 heteroatoms            independently selected from nitrogen, oxygen, or sulfur,            wherein said ring is substituted with 1-4 R^(e) groups,            wherein R^(e) is as defined above and described herein;

    -   (o) L is a bivalent C₁₋₈ saturated or unsaturated, straight or        branched, hydrocarbon chain; and Y is selected from:        -   (i) C₁₋₆ alkyl substituted with oxo, halogen, NO₂, or CN;        -   (ii) C₂₋₆ alkenyl optionally substituted with oxo, halogen,            NO₂, or CN; or        -   (iii) C₂₋₆ alkynyl optionally substituted with oxo, halogen,            NO₂, or CN; or        -   (iv) a saturated 3-4 membered heterocyclic ring having 1            heteroatom selected from oxygen or nitrogen wherein said            ring is substituted with 1-2 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (v) a saturated 5-6 membered heterocyclic ring having 1-2            heteroatom selected from oxygen or nitrogen wherein said            ring is substituted with 1-4 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (vi)

wherein each R, Q, Z, and R^(e) is as defined above and describedherein; or

-   -   -   (vii) a saturated 3-6 membered carbocyclic ring, wherein            said ring is substituted with 1-4 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (viii) a partially unsaturated 3-6 membered monocyclic ring            having 0-3 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, wherein said ring is substituted with 1-4            R^(e) groups, wherein each R^(e) is as defined above and            described herein; or        -   (ix) a partially unsaturated 3-6 membered carbocyclic ring,            wherein said ring is substituted with 1-4 R^(e) groups,            wherein each R^(e) is as defined above and described herein;        -   (x)

wherein each R^(e) is as defined above and described herein; or

-   -   -   (xi) a partially unsaturated 4-6 membered heterocyclic ring            having 1-2 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, wherein said ring is substituted with 1-4            R^(e) groups, wherein each R^(e) is as defined above and            described herein; or        -   (xii)

wherein each R and R^(e) is as defined above and described herein; or

-   -   -   (xiii) a 6-membered aromatic ring having 0-2 nitrogens            wherein said ring is substituted with 1-4 R^(e) groups,            wherein each R^(e) group is as defined above and described            herein; or        -   (xiv)

wherein each R^(e) is as defined above and described herein; or

-   -   -   (xv) a 5-membered heteroaryl ring having 1-3 heteroatoms            independently selected from nitrogen, oxygen, or sulfur,            wherein said ring is substituted with 1-3 R^(e) groups,            wherein each R^(e) group is as defined above and described            herein; or        -   (xvi)

wherein each R and R^(e) is as defined above and described herein; or

-   -   -   (xvii) an 8-10 membered bicyclic, saturated, partially            unsaturated, or aryl ring having 0-3 heteroatoms            independently selected from nitrogen, oxygen, or sulfur,            wherein said ring is substituted with 1-4 R^(e) groups,            wherein R^(e) is as defined above and described herein;

    -   (p) L is a covalent bond, —CH₂—, —NH—, —C(O)—, —CH₂NH—, —NHCH₂—,        —NHC(O)—, —NHC(O)CH₂OC(O)—, —CH₂NHC(O)—, —NHSO₂—, —NHSO₂CH₂—,        —NHC(O)CH₂OC(O)—, or —SO₂NH—; and Y is selected from:        -   (i) C₁₋₆ alkyl substituted with oxo, halogen, NO₂, or CN; or        -   (ii) C₂₋₆ alkenyl optionally substituted with oxo, halogen,            NO₂, or CN; or        -   (iii) C₂₋₆ alkynyl optionally substituted with oxo, halogen,            NO₂, or CN; or        -   (iv) a saturated 3-4 membered heterocyclic ring having 1            heteroatom selected from oxygen or nitrogen wherein said            ring is substituted with 1-2 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (v) a saturated 5-6 membered heterocyclic ring having 1-2            heteroatom selected from oxygen or nitrogen wherein said            ring is substituted with 1-4 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (vi)

wherein each R, Q, Z, and R^(e) is as defined above and describedherein; or

-   -   -   (vii) a saturated 3-6 membered carbocyclic ring, wherein            said ring is substituted with 1-4 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (viii) a partially unsaturated 3-6 membered monocyclic ring            having 0-3 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, wherein said ring is substituted with 1-4            R^(e) groups, wherein each R^(e) is as defined above and            described herein; or        -   (ix) a partially unsaturated 3-6 membered carbocyclic ring,            wherein said ring is substituted with 1-4 R^(e) groups,            wherein each R^(e) is as defined above and described herein;        -   (x)

wherein each R^(e) is as defined above and described herein; or

-   -   -   (xi) a partially unsaturated 4-6 membered heterocyclic ring            having 1-2 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, wherein said ring is substituted with 1-4            R^(e) groups, wherein each R^(e) is as defined above and            described herein; or        -   (xii)

wherein each R and R^(e) is as defined above and described herein; or

-   -   -   (xiii) a 6-membered aromatic ring having 0-2 nitrogens            wherein said ring is substituted with 1-4 R^(e) groups,            wherein each R^(e) group is as defined above and described            herein; or        -   (xiv)

wherein each R^(e) is as defined above and described herein; or

-   -   -   (xv) a 5-membered heteroaryl ring having 1-3 heteroatoms            independently selected from nitrogen, oxygen, or sulfur,            wherein said ring is substituted with 1-3 R^(e) groups,            wherein each R^(e) group is as defined above and described            herein; or        -   (xvi)

wherein each R and R^(e) is as defined above and described herein; or

-   -   -   (xvii) an 8-10 membered bicyclic, saturated, partially            unsaturated, or aryl ring having 0-3 heteroatoms            independently selected from nitrogen, oxygen, or sulfur,            wherein said ring is substituted with 1-4 R^(e) groups,            wherein R^(e) is as defined above and described herein.

In certain embodiments, the Y group of formula I is selected from thoseset forth in Table 1, below, wherein each wavy line indicates the pointof attachment to the rest of the molecule.

TABLE 1 Exemplary Y groups:

a

b

c

d

e

f

g

h

i

j

k

l

m

n

o

p

q

r

s

t

u

v

w

x

y

z

aa

bb

cc

dd

ee

ff

gg

hh

ii

jj

kk

ll

mm

nn

oo

pp

qq

rr

ss

tt

uu

vv

ww

xx

yy

zz

aaa

bbb

ccc

ddd

eee

fff

ggg

hhh

iii

jjj

kkk

lll

mmm

nnn

ooo

ppp

qqq

rrr

sss

ttt

uuu

vvv

qqq

www

xxx

yyy

zzz

aaaa

bbbb

cccc

dddd

eeee

ffff

gggg

hhhh

iiii

jjjj

kkkk

llll

mmmm

nnnn

oooo

pppp

qqqq

rrrr

ssss

tttt

uuuu

vvvv

wwww

xxxx

yyyy

zzzz

aaaaa

bbbbb

cccccwherein each R^(e) group depicted in Table 1 is independently selectedfrom halogen.

In certain embodiments, R³ is —C≡CH, —C≡CCH₂NH(isopropyl),—NHC(O)C≡CCH₂CH₃, —CH₂—C≡C—CH₃, —C≡CCH₂OH, —CH₂C(O)C≡CH, —C(O)C≡CH, or—CH₂OC(═O)C≡CH. In some embodiments, R³ is selected from —NHC(O)CH═CH₂,—NHC(O)CH═CHCH₂N(CH₃)₂, or —CH₂NHC(O)CH═CH₂.

In certain embodiments, R³ is selected from those set forth in Table 2,below, wherein each wavy line indicates the point of attachment to therest of the molecule.

TABLE 2 Exemplary R³ Groups

a

b

c

d

e

f

g

h

i

j

k

l

m

n

o

p

q

r

s

t

u

v

w

x

y

z

aa

bb

cc

dd

ee

ff

gg

hh

ii

jj

kk

ll

mm

nn

oo

pp

qq

rr

ss

tt

uu

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xxxxxxwherein each R^(e) is independently a suitable leaving group, NO₂, CN,or oxo.

In certain embodiments, one of R^(a) and R^(b) is hydrogen and the otheris —OH or —OC(O)R′. In other embodiments, R^(a) and R^(b) are takentogether to form an oxo group. In certain embodiments, one of R^(a) andR^(b) is hydrogen and the other is —OH. In certain embodiments, one ofR^(a) and R^(b) is hydrogen and the other is —OC(O)R′. In someembodiments, wherein one of R^(a) and R^(b) is —OC(O)R′, R′ is anoptionally substituted group selected from C₁₋₆ aliphatic, C₃₋₇cycloalkyl, 6-10 membered aryl, or 5-10 membered heteroaryl having 1-4heteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments, R′ is substituted C₁₋₆ aliphatic. In some embodiments,R′ is trifluoromethyl.

In certain embodiments, R¹ is an optionally substituted C₁₋₆ aliphaticgroup. In some embodiments, R¹ is optionally substituted C₁₋₃ aliphatic.In some embodiments, R¹ is n-propyl. In some embodiments, R¹ is anoptionally substituted C₃₋₇ cycloalkyl(C₁₋₃ alkyl) group. In someembodiments, R¹ is an optionally substituted C₄-₆ cycloalkyl(C₁₋₃ alkyl)group. In some embodiments, R¹ is

In certain embodiments, R² is hydrogen. In certain embodiments, R² is anoptionally substituted group selected from C₁₋₆ aliphatic or C₃₋₇cycloalkyl. In some embodiments, R² is C₃₋₇ cycloalkyl. In someembodiments, R² is cyclopropyl.

As described above, the R^(x) and R^(y) groups of formula I are takentogether to form an optionally substituted C₃₋₇ membered ring having 0-2heteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments, the R^(x) and R^(y) groups of formula I are takentogether to form an optionally substituted C₃₋₇ membered carbocyclicring. In some embodiments, the R^(x) and R^(y) groups of formula I aretaken together to form an optionally substituted cyclopropyl ring. Insome embodiments, the R^(x) and R^(y) groups of formula I are takentogether to form an optionally substituted cyclopentyl ring.

In certain embodiments, the R⁴ group of formula I is —NHC(O)NHR⁵. Insome embodiments, the R⁴ group of formula I is —NHC(O)OR⁶. In otherembodiments, the R⁴ group of formula I is

As described above, the R⁵, R⁶, and R⁷ groups of formula I areindependently optionally substituted groups selected from optionallysubstituted group selected from C₁₋₆ aliphatic, bridged bicyclic, 6-10membered aryl, 5-10 membered heteroaryl having 1-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or 4-7 memberedheterocyclyl having 1-2 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In some embodiments, R⁵ is an optionallysubstituted 5-10 membered heteroaryl having 1-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, and R⁷ is anoptionally substituted C₁₋₆ aliphatic group. In some embodiments, R⁵ is

and R⁷ is cyclohexyl.

In certain embodiments, R⁴ is —NHC(O)NHR⁵, wherein R⁵ is an optionallysubstituted group selected from optionally substituted group selectedfrom C₁₋₆ aliphatic, bridged bicyclic, 6-10 membered aryl, 5-10 memberedheteroaryl having 1-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur, or 4-7 membered heterocyclyl having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, R⁵ is optionally substituted C₁₋₆ aliphatic. In someembodiments, R⁵is t-butyl.

In certain embodiments, R⁴ is —NHC(O)OR⁶, wherein R⁶ is an optionallysubstituted group selected from optionally substituted group selectedfrom C₁₋₆ aliphatic, bridged bicyclic, 6-10 membered aryl, 5-10 memberedheteroaryl having 1-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur, or 4-7 membered heterocyclyl having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, R⁶ is optionally substituted C₁₋₆ aliphatic. In someembodiments, R⁶ is t-butyl.

In certain embodiments, the R⁵ group of formula I is an optionallysubstituted 5-10 membered heteroaryl ring having 1-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, R⁵ is an optionally substituted 6-membered heteroaryl ringhaving 1-2 nitrogens. In certain embodiments, R⁵ is

In certain embodiments, the R⁷ group of formula I is an optionallysubstituted C₁₋₆ aliphatic group. In some embodiments, R⁷ is a branchedC₁₋₅ alkyl group. In some embodiments, R⁷ is an optionally substitutedC₃₋₇ cycloalkyl group. In some embodiments, R⁷ is cyclohexyl.

In certain embodiments, R^(x) and R^(y) are taken together to form acyclopentyl ring. Thus, the present invention provides a compound offormula II-a:

or a pharmaceutically acceptable salt thereof, wherein each of R¹, R²,R³, R⁵, R⁷, R^(a), and R^(b) is as defined above for formula I anddescribed in classes and subclasses above and herein.

In some embodiments, the R¹ group of formula II-a is optionallysubstituted C₁₋₆ aliphatic. In some embodiments, R¹ is n-propyl.

In certain embodiments, the R² group of formula II-a is an optionallysubstituted C₃₋₇ cycloalkyl group. In certain embodiments, R² iscyclopropyl. In some embodiments, R² is hydrogen.

In some embodiments, the R⁵ group of formula II-a is an optionallysubstituted 5-10 membered heteroaryl group having 1-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, R⁵ is

In some embodiments, the R⁷ group of formula II-a is an optionallysubstituted C₃₋₇ cycloalkyl group. In some embodiments, R⁷ iscyclohexyl.

Exemplary R³ groups of formula II-a include those described above andherein, as well as those depicted in Table 2, above.

In certain embodiments, R^(x) and R^(y) are taken together to form anoptionally substituted cyclopropyl ring. In some embodiments, suchcompounds are of formula II-b:

or a pharmaceutically acceptable salt thereof, wherein each R¹, R², R³,R⁵, R^(a), and R^(b) is as defined in formula I and described in classesand subclasses above and herein.

In some embodiments, the R¹ group of formula II-b is optionallysubstituted C₃₋₇ cycloalkyl(C₁₋₃ alkyl). In some embodiments, R¹ is

In certain embodiments, the R² group of formula II-b is an optionallysubstituted C₃₋₇ cycloalkyl group. In certain embodiments, R² iscyclopropyl. In some embodiments, R² is hydrogen.

In some embodiments, the R⁵ group of formula II-b is an optionallysubstituted C₁₋₆ aliphatic group. In some embodiments, R⁵ is t-butyl.

Exemplary R³ groups of formula II-b include those described above andherein, as well as those depicted in Table 2, above.

In certain embodiments, R^(a) and R^(b) are taken together to form anoxo group. In certain embodiments, the present invention providescompounds of formula III-a or III-b:

or a pharmaceutically acceptable salt thereof, wherein each of R¹, R²,R³, R⁵, and R⁷ is as defined in formula I and described in classes andsubclasses above and herein.

In certain embodiments, one of R^(a) and R^(b) is hydrogen and the otheris —OC(O)R′. In some embodiments, such compounds are of formula IV-a orIV-b:

or a pharmaceutically acceptable salt thereof, wherein each of R′, R¹,R², R³, R⁵, and R⁷ is as defined in formula I and described in classesand subclasses above and herein.

In certain embodiments, the R′ group of formula IV-a and IV-b is anoptionally substituted group selected from C₁₋₆ aliphatic, C₃₋₇cycloalkyl, 6-10 membered aryl, or 5-10 membered heteroaryl having 1-4heteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments, R′ is substituted C₁₋₆ aliphatic. In some embodiments,R′ is trifluoromethyl.

In certain embodiments, one of R^(a) and R^(b) is hydrogen and the otheris —OH. In some embodiments, such compounds are of formula V-a or V-b:

or a pharmaceutically acceptable salt thereof, wherein each of R¹, R²,R³, R⁵, and R⁷ is as defined in formula I and described in classes andsubclasses above and herein.

While compounds of formulae IV-a and IV-b are depicted as havingunspecified stereochemistry at the carbon to which the ester group isattached, it will be understood that, in certain embodiments, compoundsof formulae IV-a and IV-b may be provided having either (R) or (S)stereochemistry at this position. In certain embodiments, such compoundsare of formula IV-a-1, IV-a-2, IV-b-1, or IV-b-2:

While compounds of formulae V-a and V-b, are depicted as havingunspecified stereochemistry at the carbon to which the hydroxy group isattached, it will be understood that, in certain embodiments, compoundsof formulae V-a and V-b may be provided having either (R) or (S)stereochemistry at this position. In certain embodiments, such compoundsare of formula V-a-1, V-a-2, V-b-1, or V-b-2:

Exemplary compounds of formula I are set forth in Table 3 below.

TABLE 3 Exemplary Compounds of Formula I

I-1

I-2

I-3

I-4

I-5

I-6

I-7

I-8

I-9

I-10

I-11

I-12

I-13

I-14

I-15

I-16

I-17

I-18

I-19

I-20

I-21

I-22

I-23

In certain embodiments, the present invention provides any compounddepicted in Table 3, above, or a pharmaceutically acceptable saltthereof

As defined generally above, R³ is a warhead group. Without wishing to bebound by any particular theory, it is believed that such R³ groups, i.e.warhead groups, are particularly suitable for covalently binding to akey cysteine residue in the binding domain of HCV protease. One ofordinary skill in the art will appreciate that HCV protease, and mutantsthereof, have a cysteine residue in the binding domain. In certainembodiments, compounds of the present invention have a warhead groupcharacterized in that inventive compounds may target the C159 cysteineresidue of HCV protease.

Thus, in some embodiments, R³ is characterized in that the -L-Y moietyis capable of covalently binding to a cysteine residue therebyirreversibly inhibiting the enzyme. In certain embodiments, the cysteineresidue is Cys159 of HCV protease, or a mutant thereof, where theprovided residue numbering is in accordance with Uniprot (code Q91RS4).

One of ordinary skill in the art will recognize that a variety ofwarhead groups, as defined herein, are suitable for such covalentbonding. Such R³ groups include, but are not limited to, those describedherein and depicted in Table 2, supra. This phenomenon may be determinedby performing mass spectroscopic experiments using the protocoldescribed in detail in the Exemplification, infra.

According to another aspect, the present invention provides a conjugatecomprising HCV protease, or a mutant thereof, covalently bonded to aninhibitor at Cys159. In some embodiments, the inhibitor is covalentlybonded via a linker moiety.

In certain embodiments, the present invention provides a conjugate ofthe formula Cys159-linker-inhibitor moiety. One of ordinary skill in theart will recognize that the “linker” group corresponds to a—(CH₂)_(n)-L-Y warhead group as described herein. Accordingly, incertain embodiments, the linker group is as defined for —(CH₂)_(n)-L-Ywas defined above and described in classes and subclasses herein. Itwill be appreciated, however, that the linker group is bivalent and,therefore, the corresponding —(CH₂)_(n)-L-Y group is also intended to bebivalent resulting from the reaction of the warhead with the Cys159 ofHCV protease, or a mutant thereof.

In certain embodiments, the inhibitor moiety is a compound of formula A:

wherein each of the R¹, R², R⁴, R^(a), R^(b), R^(x), and R^(y) groups offormula A is as defined for formula I above and described in classes andsubclasses herein. Thus, in certain embodiments, the present inventionprovides a conjugate of the formula:

wherein each of the R¹, R², R⁴, R^(a), R^(b), R^(x), and R^(y) groups ofthe conjugate is as defined for formula I above and described in classesand subclasses herein.

In some embodiments, R³ is characterized in that the -L-Y moiety iscapable of covalently binding to a cysteine residue thereby irreversiblyinhibiting the enzyme. In certain embodiments, the cysteine residue isCys16 of HCV protease, or a mutant thereof, where the provided residuenumbering is in accordance with Uniprot (code Q91RS4).

According to another aspect, the present invention provides a conjugatecomprising HCV protease, or a mutant thereof, covalently bonded to aninhibitor at Cys16. In some embodiments, the inhibitor is covalentlybonded via a linker moiety.

In certain embodiments, the present invention provides a conjugate ofthe formula Cys16-linker-inhibitor moiety. One of ordinary skill in theart will recognize that the “linker” group corresponds to a—(CH₂)_(n)-L-Y warhead group as described herein. Accordingly, incertain embodiments, the linker group is as defined for —(CH₂)_(n)-L-Ywas defined above and described in classes and subclasses herein. Itwill be appreciated, however, that the linker group is bivalent and,therefore, the corresponding —(CH₂)_(n)-L-Y group is also intended to bebivalent resulting from the reaction of the warhead with the Cys16 ofHCV protease, or a mutant thereof.

Thus, in certain embodiments, the present invention provides a conjugateof the formula:

wherein each of the R¹, R², R⁴, R^(a), R^(b), R^(x), and R^(y) groups ofthe conjugate is as defined for formula I above and described in classesand subclasses herein.General Methods of Providing the Present Compounds

In certain embodiments, the present compounds are generally preparedaccording to Scheme 1 set forth below.

wherein R^(3′) is selected from R³, —(CH₂)_(n)-L-H, or —(CH₂)_(n)-L-PG,each of n, L, R¹, R², and R⁵ is as defined for formula I above anddescribed in classes and subclasses herein; and PG is as describedbelow.

In one aspect, the present invention provides methods for preparingcompounds of formula I, according to the steps depicted in Scheme 1above wherein each variable is as defined and described herein and eachPG is a suitable protecting group. At step S-1, an N-protected (e.g.Boc) ester of formula a is saponified to give a carboxylic acid offormula b. At step S-2, a carboxylic acid of formula b is condensed witha compound of formula c using peptide coupling conditions to give analpha-hydroxy amide of formula d. Suitable peptide coupling conditionsare well known in the art and include those described in detail in PCTpublication number WO2002094822 (U.S. Pat. No. 6,825,347), the entiretyof which is hereby incorporated by reference. Unless otherwiseindicated, said conditions are referenced as suitable peptide couplingconditions throughout this application.

At step S-3, cleavage of the protective group (e.g. Boc removal) from analpha-hydroxy amide of formula d gives an amine of formula e, or a saltthereof. In certain embodiments, cleavage of the PG group is achieved bycontacting an alpha-hydroxy amide of formula d with a mineral or organicacid in a suitable solvent. In some embodiments, the acid ishydrochloric acid. In some embodiments, the solvent is dioxane.

At step S-4, an amine of formula e is coupled with a carboxylic acid offormula f using suitable peptide coupling conditions to an intermediatecompound of formula g.

In some embodiments, intermediate compound of formula g is converted tocompounds of formula I in steps which are described as examples herein.

In some embodiments, compounds of formula g are reacted as shown in stepS-5, wherein a compound of formula g is oxidized under suitableconditions to form a compound of formula I′. In some embodiments, thesuitable conditions comprise Dess-Martin periodinane.

As defined generally above, the PG group of formulae a, b, and d is asuitable amino protecting group. Suitable amino protecting groups arewell known in the art and include those described in detail inProtecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts,3^(rd) edition, John Wiley & Sons, 1999, the entirety of which isincorporated herein by reference. Protected amines are well known in theart and include those described in detail in Greene (1999). Suitablemono-protected amines further include, but are not limited to,aralkylamines, carbamates, allyl amines, amides, and the like. Examplesof suitable mono-protected amino moieties includet-butyloxycarbonylamino (—NHBOC), ethyloxycarbonylamino,methyloxycarbonylamino, trichloroethyloxycarbonylamino,allyloxycarbonylamino (—NHAlloc), benzyloxocarbonylamino (—NHCBZ),allylamino, benzylamino (—NHBn), fluorenylmethylcarbonyl (—NHFmoc),formamido, acetamido, chloroacetamido, dichloroacetamido,trichloroacetamido, phenylacetamido, trifluoroacetamido, benzamido,t-butyldiphenylsilyl, and the like. Suitable di-protected amines includeamines that are substituted with two substituents independently selectedfrom those described above as mono-protected amines, and further includecyclic imides, such as phthalimide, maleimide, succinimide, and thelike.

In other embodiments, the present compounds are generally preparedaccording to Scheme 1a set forth below.

wherein R³ is selected from R³, —(CH₂)_(n)-L-H, or —(CH₂)_(n)-L-PG, eachof n, L, R¹, R², and R⁶ is as defined for formula I above and describedin classes and subclasses herein; and PG is as described below.

At step S-6, cleavage of the protective group (e.g. Boc removal) from analpha-hydroxy amide of formula d, followed by coupling with a carboxylicacid of formula h using suitable peptide coupling conditions provides anintermediate compound of formula j.

In some embodiments, intermediate compound of formula j is converted tocompounds of formula I in steps which are described as examples herein.

In some embodiments, compounds of formula j are reacted as shown in stepS-7, wherein a compound of formula j is oxidized under suitableconditions to form a compound of formula I″. In some embodiments, thesuitable conditions comprise Dess-Martin periodinane.

In other embodiments, the present compounds are generally preparedaccording to Scheme 2 set forth below.

wherein R^(3′) is selected from R³, —(CH₂)_(n)-L-H, or —(CH₂)_(n)-L-PG,each of n, L, R¹, R², R⁵, R⁶ and R⁷ is as defined for formula I aboveand described in classes and subclasses herein; and PG is as describedbelow.

At step S-8, a carboxylic acid of formula k is condensed with analpha-aminoester of formula m, or a salt thereof, using suitable peptidecoupling conditions to give a carboxylic acid of formula n.

At step S-9, a carboxylic acid of formula n is condensed with analpha-aminoester of p, or a salt thereof, using suitable peptidecoupling conditions to give a carboxylic acid formula q.

At step S-10, a carboxylic acid of formula q is condensed with a prolinederivative of formula r, or a salt thereof, using suitable peptidecoupling conditions to give a compound of formula s.

In some embodiments, intermediate compound of formula s is converted tocompounds of formula I in steps which are described as examples herein.

In some embodiments, compounds of formula s are reacted as shown in stepS-11, wherein a compound of formula s is oxidized under suitableconditions to form a compound of formula I′″. In some embodiments, thesuitable conditions comprise Dess-Martin periodinane.

4. Uses, Formulation and Administration

Pharmaceutically Acceptable Compositions

According to another embodiment, the invention provides a compositioncomprising a compound of this invention or a pharmaceutically acceptablederivative thereof and a pharmaceutically acceptable carrier, adjuvant,or vehicle. The amount of compound in compositions of this invention issuch that is effective to measurably inhibit HCV protease, or a mutantthereof, in a biological sample or in a patient. In certain embodiments,the amount of compound in compositions of this invention is such that iseffective to measurably inhibit HCV protease, or a mutant thereof, in abiological sample or in a patient. In certain embodiments, a compositionof this invention is formulated for administration to a patient in needof such composition. In some embodiments, a composition of thisinvention is formulated for oral administration to a patient.

The term “patient,” as used herein, means an animal, preferably amammal, and most preferably a human.

The term “pharmaceutically acceptable carrier, adjuvant, or vehicle”refers to a non-toxic carrier, adjuvant, or vehicle that does notdestroy the pharmacological activity of the compound with which it isformulated. Pharmaceutically acceptable carriers, adjuvants or vehiclesthat may be used in the compositions of this invention include, but arenot limited to, ion exchangers, alumina, aluminum stearate, lecithin,serum proteins, such as human serum albumin, buffer substances such asphosphates, glycine, sorbic acid, potassium sorbate, partial glyceridemixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,polyethylene glycol and wool fat.

A “pharmaceutically acceptable derivative” means any non-toxic salt,ester, salt of an ester or other derivative of a compound of thisinvention that, upon administration to a recipient, is capable ofproviding, either directly or indirectly, a compound of this inventionor an inhibitorily active metabolite or residue thereof.

As used herein, the term “inhibitorily active metabolite or residuethereof” means that a metabolite or residue thereof is also an inhibitorof HCV protease, or a mutant thereof.

Compositions of the present invention may be administered orally,parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term “parenteral”as used herein includes subcutaneous, intravenous, intramuscular,intra-articular, intra-synovial, intrasternal, intrathecal,intrahepatic, intralesional and intracranial injection or infusiontechniques. Preferably, the compositions are administered orally,intraperitoneally or intravenously. Sterile injectable forms of thecompositions of this invention may be aqueous or oleaginous suspension.These suspensions may be formulated according to techniques known in theart using suitable dispersing or wetting agents and suspending agents.The sterile injectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parenterally acceptable diluent orsolvent, for example as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium.

For this purpose, any bland fixed oil may be employed includingsynthetic mono- or di-glycerides. Fatty acids, such as oleic acid andits glyceride derivatives are useful in the preparation of injectables,as are natural pharmaceutically-acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions may also contain a long-chain alcohol diluentor dispersant, such as carboxymethyl cellulose or similar dispersingagents that are commonly used in the formulation of pharmaceuticallyacceptable dosage forms including emulsions and suspensions. Othercommonly used surfactants, such as Tweens, Spans and other emulsifyingagents or bioavailability enhancers which are commonly used in themanufacture of pharmaceutically acceptable solid, liquid, or otherdosage forms may also be used for the purposes of formulation.

Pharmaceutically acceptable compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, aqueous suspensions or solutions. In thecase of tablets for oral use, carriers commonly used include lactose andcorn starch. Lubricating agents, such as magnesium stearate, are alsotypically added. For oral administration in a capsule form, usefuldiluents include lactose and dried cornstarch. When aqueous suspensionsare required for oral use, the active ingredient is combined withemulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added.

Alternatively, pharmaceutically acceptable compositions of thisinvention may be administered in the form of suppositories for rectaladministration. These can be prepared by mixing the agent with asuitable non-irritating excipient that is solid at room temperature butliquid at rectal temperature and therefore will melt in the rectum torelease the drug. Such materials include cocoa butter, beeswax andpolyethylene glycols.

Pharmaceutically acceptable compositions of this invention may also beadministered topically, especially when the target of treatment includesareas or organs readily accessible by topical application, includingdiseases of the eye, the skin, or the lower intestinal tract. Suitabletopical formulations are readily prepared for each of these areas ororgans.

Topical application for the lower intestinal tract can be effected in arectal suppository formulation (see above) or in a suitable enemaformulation. Topically-transdermal patches may also be used.

For topical applications, provided pharmaceutically acceptablecompositions may be formulated in a suitable ointment containing theactive component suspended or dissolved in one or more carriers.Carriers for topical administration of compounds of this inventioninclude, but are not limited to, mineral oil, liquid petrolatum, whitepetrolatum, propylene glycol, polyoxyethylene, polyoxypropylenecompound, emulsifying wax and water. Alternatively, providedpharmaceutically acceptable compositions can be formulated in a suitablelotion or cream containing the active components suspended or dissolvedin one or more pharmaceutically acceptable carriers. Suitable carriersinclude, but are not limited to, mineral oil, sorbitan monostearate,polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol,benzyl alcohol and water.

For ophthalmic use, provided pharmaceutically acceptable compositionsmay be formulated as micronized suspensions in isotonic, pH adjustedsterile saline, or, preferably, as solutions in isotonic, pH adjustedsterile saline, either with or without a preservative such asbenzylalkonium chloride. Alternatively, for ophthalmic uses, thepharmaceutically acceptable compositions may be formulated in anointment such as petrolatum.

Pharmaceutically acceptable compositions of this invention may also beadministered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other conventional solubilizingor dispersing agents.

Most preferably, pharmaceutically acceptable compositions of thisinvention are formulated for oral administration. Such formulations maybe administered with or without food. In some embodiments,pharmaceutically acceptable compositions of this invention areadministered without food. In other embodiments, pharmaceuticallyacceptable compositions of this invention are administered with food.

The amount of compounds of the present invention that may be combinedwith the carrier materials to produce a composition in a single dosageform will vary depending upon the host treated, the particular mode ofadministration. Preferably, provided compositions should be formulatedso that a dosage of between 0.01-100 mg/kg body weight/day of theinhibitor can be administered to a patient receiving these compositions.

It should also be understood that a specific dosage and treatmentregimen for any particular patient will depend upon a variety offactors, including the activity of the specific compound employed, theage, body weight, general health, sex, diet, time of administration,rate of excretion, drug combination, and the judgment of the treatingphysician and the severity of the particular disease being treated. Theamount of a compound of the present invention in the composition willalso depend upon the particular compound in the composition.

Uses of Compounds and Pharmaceutically Acceptable Compositions

Compounds and compositions described herein are generally useful for theinhibition of HCV protease activity and/or the activity of a mutantthereof. Thus, provided compounds are useful for treating non-A, non-Bhepatitis, including hepatitis C.

HCV is an extremely variable virus that forms polymorphic swarms ofvariants within the host. Worldwide, six different genotypes have nowbeen defined (Simmonds et al., Hepatology, Vol. 42, No. 4, 2005). Thesegenotypes have been further classified into more closely related,genetically distinct subtypes. Comparative sequence portions, known asconsensus sequences, are set forth in Table 3a, below. HCV genotypes andsubtypes are distributed differently in different parts of the world,and certain genotypes predominate in certain areas. Genotypes 1-3 arewidely distributed throughout the world. Subtype 1a is prevalent inNorth and South America, Europe, and Australia. Subtype 1b is common inNorth America and Europe, and is also found in parts of Asia. Genotype 2is present in most developed countries, but is less common than genotype1 (http://www.hcvadvocate.org/hepatitis/factsheets_pdf/genotype_FS.pdf).Other genotypes are prevalent in ex-US patient populations and aretherefore important targets.

Notably, a cysteine located at amino acid position 159 in genotype 1b isconserved in all genotypes and subtypes of HCV NS3 sequenced to date,although the amino acid position may be different in other genotypes andsubtypes. Targeting this cysteine residue with irreversible inhibitorsshould enable the development of agents which are effective againstmultiple HCV genotypes.

As described herein, the present invention provides irreversibleinhibitors of one or more HCV protease genotypes, and variants thereof.Such compounds, comprising a warhead group designated as R³, includethose of formulae I, II-a, II-b, III-a, III-b, IV-a, IV-b, V-a, V-b,IV-a-1, IV-a-2, IV-b-1, IV-b-2, V-a-1, V-a-2, V-b-1, and V-b-2, asdescribed herein. In some embodiments, R³ is characterized in that the-L-Y moiety is capable of covalently binding to a cysteine residuethereby irreversibly inhibiting the enzyme. Without wishing to be boundby any particular theory, it is believed that such R³ groups, i.e.warhead groups, are particularly suitable for covalently binding to akey cysteine residue in the binding domain of one or more HCV proteasegenotypes or variants thereof. In some embodiments, one or moregenotypes inhibited by compounds of the present invention include 1a,1b, 2a, and 3a. In certain embodiments, one or more such variantsinclude A156T, A156S, D168V, D168A, and R155K.

One of ordinary skill in the art will appreciate that HCV proteasegenotypes and variants thereof have one or more cysteine residues nearthe binding domain. Without wishing to be bound by any particulartheory, it is believed that proximity of a warhead group to the cysteineof interest facilitates covalent modification of that cysteine by thewarhead group. In some embodiments, the cysteine residue of interest isCys159 of HCV protease subtype 1b, or a variant thereof, where theprovided residue numbering is in accordance with Uniprot (code Q91RS4).Cysteine residues of other HCV protease genotypes and subtypes suitablefor covalent modification by irreversible inhibitors of the presentinvention include those summarized in Table 3a, below, where the boldand underlined “C” refers to a cysteine residue conserved at anequivalent position to Cys159 of HCV protease subtype 1b.

TABLE 3a HCV Representative genotype/ Sequence Sequence subtypePortion^(a) Patient ID Identifier 1a GHAVGLFRAAV C TRGVAKAV_.H77.NC_004102 SEQ ID NO: 1 1a GHAVGIFRAAV C TRGVAKAVCH.BID-V271.EU482858 SEQ ID NO: 2 1a GHAVGIFRAAV C TRGVAKAVDE.BID-V25.EU482831 SEQ ID NO: 3 1a GHAVGLFRAAV C TRGVAKAVUS.H77-H21.AF011753 SEQ ID NO: 4 1b GHAVGIFRAAV C TRGVAKAVAU.HCV-A.AJ000009 SEQ ID NO: 5 1b GHVVGIFRAAV C TRGVAKAVCH.BID-V272.EU482859 SEQ ID NO: 6 1b GHAVGIFRAAV C TRGVAKAVJP.HCV-BK.M58335 SEQ ID NO: 7 1c GHAVGIFRAAV C TRGVAKAV ID.HC-G9.D14853SEQ ID NO: 8 1c GHVAGIFRAAV C TRGVAKAV IN.AY051292.AY051292 SEQ ID NO: 92a GHAVGIFRAAV C SRGVAKSI JP.AY746460.AY746460 SEQ ID NO: 10 2aGHAVGIFRAAV C SRGVAKSI JP.JCH-6.AB047645 SEQ ID NO: 11 2a GHAVGIFRAAV CSRGVAKSI _.G2AK1.AF169003 SEQ ID NO: 12 2b GHAVGLFRAAV C ARGVAKSIJP.HC-J8.D10988 SEQ ID NO: 13 2b GHAVGLFRAAV C ARGVAKSIJP.MD2b1-2.AY232731 SEQ ID NO: 14 2c GHAVGIFRAAV C SRGVAKSI_.BEBE1.D50409 SEQ ID NO: 15 2i AHAVGIFRAAV C SRGVAKSI VN.D54.DQ155561SEQ ID NO: 16 2k GHAVGIFRAAI C TRGAAKSI MD.VAT96.AB031663 SEQ ID NO: 173a GHVAGIFRAAV C TRGVAKAL CH.452.DQ437509 SEQ ID NO: 18 3a GHVAGIFRAAV CTRGVAKAL DE.HCVCENS1.X76918 SEQ ID NO: 19 3a GHVAGIFRAAV C TRGVAKALID.ps23.EU315121 SEQ ID NO: 20 3b GHVMGIFIAVV C TRGVAKALIN.RG416.DQ284965 SEQ ID NO: 21 3b GHVVGIFRAAV C TRGVAKALJP.HCV-Tr.D49374 SEQ ID NO: 22 3k GHVAGIFRAAV C TRGVAKAL ID.JK049.D63821SEQ ID NO: 23 4a GHAAGIFRAAV C TRGVAKAV EG.Eg9.DQ988077 SEQ ID NO: 24 4aGHAAGLFRAAV C TRGVAKAV _.01-09.DQ418782 SEQ ID NO: 25 4a GHAAGLFRAAV CTRGVAKAV _.F753.DQ418787 SEQ ID NO: 26 4d GHAAGIFRAAV C TRGVAKAV_.03-18.DQ418786 SEQ ID NO: 27 4d GHAAGIFRAAV C TRGVAKTV _.24.DQ516083SEQ ID NO: 28 4f GHAVGIFRAAV C TRGVAKAV FR.IFBT84.EF589160 SEQ ID NO: 294f GHAVGIFRAAV C TRGVAKAV FR.IFBT88.EF589161 SEQ ID NO: 30 5aGHVVGVFRAAV C TRGVAKAL GB.EUH1480.Y13184 SEQ ID NO: 31 5a GHVVGVFRAAV CTRGVAKAL ZA.SA13.AF064490 SEQ ID NO: 32 6a GHVVGLFRAAV C TRGVAKSLHK.6a74.DQ480524 SEQ ID NO: 33 6a GHVVGLFRAAV C TRGVAKSLHK.6a77.DQ480512 SEQ ID NO: 34 6a GHVVGLFRAAV C TRGVAKSL HK.EUHK2.Y12083SEQ ID NO: 35 6b GHVVGLFRAAV C TRGVAKAL _.Th580.NC_009827 SEQ ID NO: 366c GHVVGLFRAAV C TRGVAKAL TH.Th846.EF424629 SEQ ID NO: 37 6d DHVVGLFRAAVC TRGVAKAL VN.VN235.D84263 SEQ ID NO: 38 6e GHVVGLFRAAV C TRGVAKAICN.GX004.DQ314805 SEQ ID NO: 39 6f GHAVGIFRAAV C TRGVAKAITH.C-0044.DQ835760 SEQ ID NO: 40 6f GHAVGIFRAAV C TRGVAKAITH.C-0046.DQ835764 SEQ ID NO: 41 6g GHVVGLFRAAV C TRGVAKALHK.HK6554.DQ314806 SEQ ID NO: 42 6g GHVVGLFRAAV C TRGVAKALID.JK046.D63822 SEQ ID NO: 43 6h GHVAGIFRAAV C TRGVAKSL VN.VN004.D84265SEQ ID NO: 44 6i GHVAGIFRAAV C TRGVAKSL TH.C-0159.DQ835762 SEQ ID NO: 456j GHVAGIFRAAV C TRGVAKSL TH.C-0667.DQ835761 SEQ ID NO: 46 6jGHVAGIFRAAV C TRGVAKSL TH.Th553.DQ835769 SEQ ID NO: 47 6k GHVAGIFRAAV CTRGVAKSL CN.KM41.DQ278893 SEQ ID NO: 48 6k GHVAGIFRAAV C TRGVAKSLCN.KM45.DQ278891 SEQ ID NO: 49 6k GHVAGIFRAAV C TRGVAKSL VN.VN405.D84264SEQ ID NO: 50 6l GHVAGIFRAAV C TRGVAKSL US.537796.EF424628 SEQ ID NO: 516m GHAVGVFRAAV C TRGVAKSL TH.C-0185.DQ835765 SEQ ID NO: 52 6mGHAVGVFRAAV C TRGVAKSL TH.C-0208.DQ835763 SEQ ID NO: 53 6n GHVVGIFRAAV CTRGVAKSL CN.KM42.DQ278894 SEQ ID NO: 54 6n GHVVGIFRAAV C TRGVAKSLTH.D86/93.DQ835768 SEQ ID NO: 55 6o GHAVGLFRAAV C TRGVAKAICA.QC227.EF424627 SEQ ID NO: 56 6p GHVVGLFRAAV C TRGVAKAICA.QC216.EF424626 SEQ ID NO: 57 6q GHAVGLFRAAV C TRGVAKAICA.QC99.EF424625 SEQ ID NO: 58 6t GHVVGLFRAAV C TRGVAKAIVN.TV241.EF632069 SEQ ID NO: 59 6t GHVVGLFRAAV C TRGVAKAIVN.TV249.EF632070 SEQ ID NO: 60 6t GHVVGLFRAAV C TRGVAKAIVN.VT21.EF632071 SEQ ID NO: 61 7a SHCVGIFRAAV C TRGVAKAVCA.QC69.EF108306 SEQ ID NO: 62 ^(a)It will be appreciated by one ofordinary skill in the art that every virus is prone to mutation andsubject to polymorphisms, and any genotype consensus sequences describedherein are representative of a given genotype or subtype. Suchrepresentative consensus sequences are available athttp://hcv.lanl.gov/content/sequence/NEWALIGN/align.html.

Drug resistance is emerging as a significant challenge for targetedtherapies. For example, drug resistance has been reported for HCVprotease inhibitors in development. Such compounds include BILN 2061 andVX-950 (also known as telaprevir), developed by Boehringer Ingelheim andVertex Pharmaceuticals, respectively. The structures of BILN 2061 andVX-950 are depicted below.

In fact, a recent article published by Vertex Pharmaceuticals, entitled,“In Vitro Resistance Studies of Hepatitis C Virus Serine Protease,”squarely addresses the problem of mutant resistance observed with VX-950and BILN 2061. See Lin et al., The Journal of Biological Chemistry, Vol.279, No. 17, Issue of April 23, pp. 17508-17514, 2004. This articleconcludes that “future hepatitis C therapy involving small moleculeinhibitors of HCV enzymes might require multidrug combination, as in thecase of the current HIV treatments.” See page 17513, last paragraph.

Resistance to specific antiviral drugs is a major factor limiting theefficacy of therapies against many retroviruses or RNA viruses. Theerror-prone nature of these viruses allows for the development ofmutations that afford resistance to currently available drugs or drugsundergoing clinical testing. The resistance problem is a critical hurdlefaced in drug development of new HCV-specific inhibitors to treat HCVpatients.

A recent in vitro resistance study using two HCV NS3•4A proteaseinhibitors, VX-950 and BILN 2061, found that resistance mutationsselected against either inhibitor resulted in a significant reduction insusceptibility to the inhibitor itself. However, the primary resistancemutations against BILN 2061 were fully susceptible to VX-950, and themajor resistance mutation against VX-950 remained sensitive to BILN 2061(Lin et al., Jour. Biol. Chem. 279(17): 17508-14, 2004).

In the ensuing Examples, provided compounds of the present inventionhave an HCV activity profile reflecting inhibition of several HCVprotease mutants.

As used herein, the term “clinical drug resistance” refers to the lossof susceptibility of a drug target to drug treatment as a consequence ofmutations in the drug target

As used herein, the term “resistance” refers to changes in the wild-typenucleic acid sequence coding a target protein, and/or the proteinsequence of the target, which changes decrease or abolish the inhibitoryeffect of the inhibitor on the target protein.

Examples of proteases that are inhibited by the compounds andcompositions described herein and against which the methods describedherein are useful include NS3, NS3•4A, or a mutant thereof.

The activity of a compound utilized in this invention as an inhibitor ofNS3, NS3•4A, or a mutant thereof, may be assayed in vitro, in vivo or ina cell line. In vitro assays include assays that determine inhibition ofeither the serine protease activity and/or the subsequent functionalconsequences, or ATPase activity of activated NS3, NS3•4A, or a mutantthereof. Alternate in vitro assays quantitate the ability of theinhibitor to bind to NS3 or NS3•4A. Inhibitor binding may be measured byradiolabelling the inhibitor prior to binding, isolating theinhibitor/NS3 or inhibitor/NS3•4A complex and determining the amount ofradiolabel bound. Alternatively, inhibitor binding may be determined byrunning a competition experiment where new inhibitors are incubated withNS3 or NS3•4A bound to known radioligands. Detailed conditions forassaying a compound utilized in this invention as an inhibitor of NS3 orNS3•4A, or a mutant thereof, are set forth in the Examples below.

Serine proteases are a large family of proteolytic enzymes that cleavepeptide bonds in proteins. The serine protease family includes thedigestive enzymes chymotrypsin, trypsin, and elastase, and proteasesinvolved in blood clotting. Serine proteases possess a characteristic“catalytic triad” comprising serine, aspartic acid, and histidine, thattogether function to activate serine to form a covalent bond with theenzyme substrate, thereby hydrolyzing a peptide bond. In addition tothose stated above, serine proteases participate in a variety offunctions including immunity and inflammation.

As used herein, the terms “treatment,” “treat,” and “treating” refer toreversing, alleviating, delaying the onset of, or inhibiting theprogress of a disease or disorder, or one or more symptoms thereof, asdescribed herein. In some embodiments, treatment may be administeredafter one or more symptoms have developed. In other embodiments,treatment may be administered in the absence of symptoms. For example,treatment may be administered to a susceptible individual prior to theonset of symptoms (e.g., in light of a history of symptoms and/or inlight of genetic or other susceptibility factors). Treatment may also becontinued after symptoms have resolved, for example to prevent or delaytheir recurrence.

The compounds and compositions, according to the method of the presentinvention, may be administered using any amount and any route ofadministration effective for treating or lessening the severity ofcancer, an autoimmune disorder, a neurodegenerative or neurologicaldisorder, schizophrenia, a bone-related disorder, liver disease, or acardiac disorder. The exact amount required will vary from subject tosubject, depending on the species, age, and general condition of thesubject, the severity of the infection, the particular agent, its modeof administration, and the like. The compounds of the invention arepreferably formulated in dosage unit form for ease of administration anduniformity of dosage. The expression “dosage unit form” as used hereinrefers to a physically discrete unit of agent appropriate for thepatient to be treated. It will be understood, however, that the totaldaily usage of the compounds and compositions of the present inventionwill be decided by the attending physician within the scope of soundmedical judgment. The specific effective dose level for any particularpatient or organism will depend upon a variety of factors including thedisorder being treated and the severity of the disorder; the activity ofthe specific compound employed; the specific composition employed; theage, body weight, general health, sex and diet of the patient; the timeof administration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed, andlike factors well known in the medical arts. The term “patient”, as usedherein, means an animal, preferably a mammal, and most preferably ahuman.

The pharmaceutically acceptable compositions of this invention can beadministered to humans and other animals orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments, or drops), bucally, as an oral or nasal spray, orthe like, depending on the severity of the infection being treated. Incertain embodiments, the compounds of the invention may be administeredorally or parenterally at dosage levels of about 0.01 mg/kg to about 50mg/kg and preferably from about 1 mg/kg to about 25 mg/kg, of subjectbody weight per day, one or more times a day, to obtain the desiredtherapeutic effect.

In some embodiments, a provided composition is administered to a patientin need thereof once daily. Without wishing to be bound by anyparticular theory, it is believed that prolonged duration of action ofan irreversible inhibitor of HCV NS3 protease is particularlyadvantageous for once daily administration to a patient in need thereoffor the treatment of a disorder associated with HCV NS3 protease. Incertain embodiments, a provided composition is administered to a patientin need thereof at least once daily. In other embodiments, a providedcomposition is administered to a patient in need thereof twice daily,three times daily, or four times daily.

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active compounds,the liquid dosage forms may contain inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof. Besides inert diluents,the oral compositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of a compound of the present invention,it is often desirable to slow the absorption of the compound fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the compound thendepends upon its rate of dissolution that, in turn, may depend uponcrystal size and crystalline form. Alternatively, delayed absorption ofa parenterally administered compound form is accomplished by dissolvingor suspending the compound in an oil vehicle. Injectable depot forms aremade by forming microencapsule matrices of the compound in biodegradablepolymers such as polylactide-polyglycolide. Depending upon the ratio ofcompound to polymer and the nature of the particular polymer employed,the rate of compound release can be controlled. Examples of otherbiodegradable polymers include poly(orthoesters) and poly(anhydrides).Depot injectable formulations are also prepared by entrapping thecompound in liposomes or microemulsions that are compatible with bodytissues.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar—agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes. Solid compositions of a similartype may also be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polethylene glycols and the like.

The active compounds can also be in micro-encapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents. They may optionally contain opacifying agents and canalso be of a composition that they release the active ingredient(s)only, or preferentially, in a certain part of the intestinal tract,optionally, in a delayed manner. Examples of embedding compositions thatcan be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, ear drops, and eye drops are also contemplatedas being within the scope of this invention. Additionally, the presentinvention contemplates the use of transdermal patches, which have theadded advantage of providing controlled delivery of a compound to thebody. Such dosage forms can be made by dissolving or dispensing thecompound in the proper medium. Absorption enhancers can also be used toincrease the flux of the compound across the skin. The rate can becontrolled by either providing a rate controlling membrane or bydispersing the compound in a polymer matrix or gel.

According to one embodiment, the invention relates to a method ofinhibiting serine protease activity in a biological sample comprisingthe step of contacting said biological sample with a compound of thisinvention, or a composition comprising said compound.

According to another embodiment, the invention relates to a method ofinhibiting HCV protease, or a mutant thereof, activity in a biologicalsample comprising the step of contacting said biological sample with acompound of this invention, or a composition comprising said compound.In certain embodiments, the invention relates to a method ofirreversibly inhibiting HCV protease, or a mutant thereof, activity in abiological sample comprising the step of contacting said biologicalsample with a compound of this invention, or a composition comprisingsaid compound.

The term “biological sample”, as used herein, includes, withoutlimitation, cell cultures or extracts thereof; biopsied materialobtained from a mammal or extracts thereof, and blood, saliva, urine,feces, semen, tears, or other body fluids or extracts thereof.

Inhibition of HCV protease, or a mutant thereof, activity in abiological sample is useful for a variety of purposes that are known toone of skill in the art. Examples of such purposes include, but are notlimited to, blood transfusion, organ-transplantation, biologicalspecimen storage, and biological assays.

Another embodiment of the present invention relates to a method ofinhibiting HCV protease, or a mutant thereof, activity in a patientcomprising the step of administering to said patient a compound of thepresent invention, or a composition comprising said compound.

According to another embodiment, the invention relates to a method ofinhibiting HCV protease, or a mutant thereof, activity in a patientcomprising the step of administering to said patient a compound of thepresent invention, or a composition comprising said compound. Accordingto certain embodiments, the invention relates to a method ofirreversibly inhibiting HCV protease, or a mutant thereof, activity in apatient comprising the step of administering to said patient a compoundof the present invention, or a composition comprising said compound. Inother embodiments, the present invention provides a method for treatinga disorder mediated by HCV protease, or a mutant thereof, in a patientin need thereof, comprising the step of administering to said patient acompound according to the present invention or pharmaceuticallyacceptable composition thereof. Such disorders are described in detailherein.

Depending upon the particular condition, or disease, to be treated,additional therapeutic agents, which are normally administered to treatthat condition, may be administered in combination with compounds andcompositions of this invention. As used herein, additional therapeuticagents that are normally administered to treat a particular disease, orcondition, are known as “appropriate for the disease, or condition,being treated”.

In certain embodiments, a provided compound, or composition thereof, isadministered in combination with another inhibitor of HCV protease, or avariant thereof. In some embodiments, a provided compound, orcomposition thereof, is administered in combination with anotherantiviral agent. Such antiviral agents include, but are not limited to,immunomodulatory agents, such as α-, β-, and γ-interferons, pegylatedderivatized interferon-α compounds, and thymosin; other anti-viralagents, such as ribavirin, amantadine, and telbivudine; other inhibitorsof hepatitis C proteases (NS2-NS3 inhibitors and NS3-NS4A inhibitors,e.g. BILN 2061 and VX-950); inhibitors of other targets in the HCV lifecycle, including helicase and polymerase inhibitors; inhibitors ofinternal ribosome entry; broad-spectrum viral inhibitors, such as IMPDHinhibitors (e.g., mycophenolic acid and derivatives thereof); orcombinations of any of the above.

In certain embodiments, a combination of 2 or more antiviral agents maybe administered. In certain embodiments, a combination of 3 or moreantiviral agents may be administered. In some embodiments, the antiviralagents are selected from ribavirin or interferon. In other embodiments,the antiviral agent is α-interferon.

Other examples of agents the inhibitors of this invention may also becombined with include, without limitation: treatments for Alzheimer'sDisease such as Aricept® and Excelon®; treatments for HIV such asritonavir; treatments for Parkinson's Disease such as L-DOPA/carbidopa,entacapone, ropinrole, pramipexole, bromocriptine, pergolide,trihexephendyl, and amantadine; agents for treating Multiple Sclerosis(MS) such as beta interferon (e.g., Avonex® and Rebif®), Copaxone®, andmitoxantrone; treatments for asthma such as albuterol and Singulair®;agents for treating schizophrenia such as zyprexa, risperdal, seroquel,and haloperidol; anti-inflammatory agents such as corticosteroids, TNFblockers, IL-1 RA, azathioprine, cyclophosphamide, and sulfasalazine;immunomodulatory and immunosuppressive agents such as cyclosporin,tacrolimus, rapamycin, mycophenolate mofetil, interferons,corticosteroids, cyclophophamide, azathioprine, and sulfasalazine;neurotrophic factors such as acetylcholinesterase inhibitors, MAOinhibitors, interferons, anti-convulsants, ion channel blockers,riluzole, and anti-Parkinsonian agents; agents for treatingcardiovascular disease such as beta-blockers, ACE inhibitors, diuretics,nitrates, calcium channel blockers, and statins; agents for treatingliver disease such as corticosteroids, cholestyramine, interferons, andanti-viral agents; agents for treating blood disorders such ascorticosteroids, anti-leukemic agents, and growth factors; agents thatprolong or improve pharmacokinetics such as cytochrome P450 inhibitors(i.e., inhibitors of metabolic breakdown) and CYP3A4 inhibitors (e.g.,ketokenozole and ritonavir), and agents for treating immunodeficiencydisorders such as gamma globulin.

In certain embodiments, compounds of the present invention, or apharmaceutically acceptable composition thereof, are administered incombination with a monoclonal antibody or an siRNA therapeutic.

Those additional agents may be administered separately from an inventivecompound-containing composition, as part of a multiple dosage regimen.Alternatively, those agents may be part of a single dosage form, mixedtogether with a compound of this invention in a single composition. Ifadministered as part of a multiple dosage regime, the two active agentsmay be submitted simultaneously, sequentially or within a period of timefrom one another normally within five hours from one another.

As used herein, the term “combination,” “combined,” and related termsrefers to the simultaneous or sequential administration of therapeuticagents in accordance with this invention. For example, a compound of thepresent invention may be administered with another therapeutic agentsimultaneously or sequentially in separate unit dosage forms or togetherin a single unit dosage form. Accordingly, the present inventionprovides a single unit dosage form comprising a compound of formula I,an additional therapeutic agent, and a pharmaceutically acceptablecarrier, adjuvant, or vehicle.

The amount of both, an inventive compound and additional therapeuticagent (in those compositions which comprise an additional therapeuticagent as described above) that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. Preferably,compositions of this invention should be formulated so that a dosage ofbetween 0.01-100 mg/kg body weight/day of an inventive can beadministered.

In those compositions which comprise an additional therapeutic agent,that additional therapeutic agent and the compound of this invention mayact synergistically. Therefore, the amount of additional therapeuticagent in such compositions will be less than that required in amonotherapy utilizing only that therapeutic agent. In such compositionsa dosage of between 0.01-100 mg/kg body weight/day of the additionaltherapeutic agent can be administered.

The amount of additional therapeutic agent present in the compositionsof this invention will be no more than the amount that would normally beadministered in a composition comprising that therapeutic agent as theonly active agent. Preferably the amount of additional therapeutic agentin the presently disclosed compositions will range from about 50% to100% of the amount normally present in a composition comprising thatagent as the only therapeutically active agent.

EXEMPLIFICATION

As depicted in the Examples below, in certain exemplary embodiments,compounds are prepared according to the following general procedures. Itwill be appreciated that, although the general methods depict thesynthesis of certain compounds of the present invention, the followinggeneral methods, and other methods known to one of ordinary skill in theart, can be applied to all compounds and subclasses and species of eachof these compounds, as described herein.

Compound numbers utilized in the Examples, below, correspond to compoundnumbers set forth in Table 3, supra.

Example 1

(1R,2,5S)-3-((S)-3-acrylamido-2-(3-tert-butylureido)propanoyl)-N-(4-amino-1-cyclobutyl-3,4-dioxobutan-2-yl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide[I-1]

The title compound was prepared according to the steps and intermediatesas described below.

Step 1a: Intermediate 1a

To a solution of (1R,2S,5S)-3-tert-butyl 2-methyl6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2,3-dicarboxylate (0.30 g, 1.1mmol) in 4 mL THF/MeOH (1:1) was added 1 N aqueous LiOH solution (2.0mL). After stirring at r.t. for 10 hours, the reaction mixture wasneutralized with 1.0 N HCl. The organic solvents were evaporated undervacuum, and the remaining aqueous phase was acidified to pH˜3 using 1.0N HCl and was extracted with EtOAc. The organic layer was washed withbrine, and was dried over anhydrous magnesium sulfate. After removal ofsolvent, 0.28 g of Intermediate 1a was obtained: MS m/z: 254.2 (ES−).

Step 1b: Intermediate 1b

To a solution of the product of step 1a (0.28 g, 1.0 mmol) and3-amino-4-cyclobutyl-2-hydroxybutanamide (0.27 g, 1.3 mmol) in 10.0 mlof anhydrous acetonitrile was added HATU (0.45 g, 1.2 mmol) and DIEA(0.5 ml, 3.0 mmol) at r.t. under stirring. TLC analysis indicatedcompletion of the coupling reaction had occurred after 10 hours. A 50-mlportion of EtOAc was added in and the mixture was washed with aqueousNaHCO₃ and brine. The organic layer was separated and was dried overNa₂SO₄. After removal of solvent, the crude product was subject tochromatography on silica gel (eluents: EtOAc/hexane). A total of 0.4 gof the title compound was obtained (88%). MS m/z: 432.2 (ES+, M+Na).

Step 1c: Intermediate 1c

The product from step 1b (0.40 g, 1.0 mmol) was dissolved in 5 mL 4 NHCl in dioxane. The mixture was stirred at r.t. for 1 hour. Afterremoval of solvents, a 10-mL portion of DCM was poured in followed byevaporation to dryness. This process of DCM addition followed byevaporation was repeated four times to give a residue solid which wasused directly for the next step: MS m/z: 310.1 (M+H⁺).

Step 1d: Intermediate 1d

To a solution of the product from step 1c (0.10 g, 0.28 mmol) and(S)-3-(tert-butoxycarbonylamino)-2-(3-tert-butylureido)propanoic acid(0.10 g, 0.33 mmol) in 3.0 mL of anhydrous acetonitrile was added HATU(125 mg, 0.33 mmol) and DIEA (0.17 mL, 1.0 mmol) at r.t. under stirring.After one hour, 15 mL of EtOAc was added in and the mixture was washedwith aqueous NaHCO₃ and brine. The organic layer was separated and wasdried over Na₂SO₄. After removal of solvent, the crude product wassubject to chromatography on silica gel (eluents: EtOAc/hexane) toafford 103 mg of the title compound (60%). MS m/z: 595.2 (M+H⁺).

Step 1e: Intermediate 1e

The product from step 1d (75 mg, 0.12 mmol) was dissolved in 3 mL of 4 NHCl in dixoxane and the reaction was stirred for 1 hour at RT. Afterremoval of solvents, a 3-mL portion of DCM was poured in followed byevaporation to dryness. This process of DCM addition followed byevaporation was repeated three times to give a light brown solid and wasused directly for the next step. MS m/z: 495.2 (M+H⁺).

Step 1f: Intermediate 1f

Acrylic acid (13.6 mg, 0.19 mmol) was coupled with the product from step1e with HATU (65 mg, 0.17 mmol) following the procedure described instep 1b to afford the title compound (60 mg, crude). MS m/z: 549.3(M+H⁺).

Step 1g:(1R,2S,5S)-3-((S)-3-acrylamido-2-(3-tert-butylureido)propanoyl)-N-(4-amino-1-cyclobutyl-3,4-dioxobutan-2-yl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide

The crude product from step 1f (60 mg, 0.11 mmol) was dissolved in 5 mlof dichloromethane followed by the addition of the Dess-Martinperiodinane (60 mg, 0.15 mmol). The resulting solution was stirred for 1h at room temperature. The solvent was then removed and the residue wassubject to chromatography on silica gel (eluents: EtOAc/Heptanes) toprovide 13 mg of the title compound. MS m/z: 547.2 (M+H⁺).

In similar fashion using the product of step 1e (Intermediate 1e),coupling with vinylsulfonylchloride followed by oxidation using theprocedures described in step 1f and 1g, the following compound can beprepared:

Following the procedures described in example 1, using(S)-4-(tert-butoxycarbonylamino)-2-(3-tert-butylureido)butanoic acid tocouple with intermediate 1c in step 1d, the following compound can beprepared:

In similar fashion, using(S)-4-(tert-butoxycarbonylamino)-2-(3-tert-butylureido)butanoic acid tocouple with intermediate 1c in step 1d, de-Boc following step 1e, thencoupling with vinylsulfonylchloride followed by oxidation using theprocedures described in step 1f and 1g, the following compound can beprepared:

Following the procedures described in example 1, using hydroxylprotected (S)-2-(3-tert-butylureido)-3-hydroxypropanoic acid to couplewith intermediate 1c in step 1d, and using crotyl chloride in step 1f,the following compound can be prepared:

Example 2

tert-butyl(S)-1-((1R,2S,5S)-2-(4-amino-1-cyclobutyl-3,4-dioxobutan-2-ylcarbamoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexan-3-yl)-7-methyl-1,5-dioxooct-6-en-2-ylcarbamate[I-6]

The title compound was prepared according to the steps and intermediatesas described below.

Step 2a: Intermediate 2a

To a solution of N-Boc-pyroglutamic acid (0.23 g 1.0 mmol) in 10.0 mL ofanhydrous THF was added 2-methylprop-1-enyl)magnesium bromide (0.5 M inTHF, 5 mL, 2.5 mmol) at −78° C. slowly. The reaction mixture was stirredfor 1 h at −78° C. 1 N HCl (2.5 ml) aqueous solution was added and themixture was slowly warmed up to RT. The pH was adjusted to ˜3-4 with 1 NHCl. The THF was then removed under vacuum and the remaining aqueous wasextracted by DCM (3×15 ml). The organic layer was dried over Na₂SO₄,filterd and the solvent was removed to provide the crude product.

Step 2b: Intermediate 2b

The title compound was made by coupling Intermediate 1c from Example 1and Intermediate 2a using HATU following the coupling reaction describedfor Intermediate 1b in Example 1. A total of 100 mg of the titlecompound (crude) was obtained from 120 mg of Intermediate 1c. MS m/z:599.3 (M+Na⁺).

Step 2c: tert-butyl(S)-1-((1R,2S,5S)-2-(4-amino-1-cyclobutyl-3,4-dioxobutan-2-ylcarbamoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexan-3-yl)-7-methyl-1,5-dioxooct-6-en-2-ylcarbamate

The crude product from step 2b (100 mg, 0.15 mmol) was dissolved in 5 mLof dichloromethane followed by the addition of the Dess-Martinperiodinane (150 mg, 0.36 mmol). The resulting solution was stirred for1 h at room temperature. The solvent was then removed and the residuewas subject to chromatography on silica gel (eluents: EtOAc/Heptanes) toprovide 60 mg of the title compound. MS m/z: 575.3 (M+H⁺).

Starting from the Intermediate 1c, by coupling with the appropriateintermediates made similarly as described in Step 2a, the followingcompounds are prepared:

Example 3

(1S,3aR,6aS)-2-((S)-3-acrylamido-2-((S)-2-cyclohexyl-2-(pyrazine-2-carboxamido)acetamido)propanoyl)-N-((S)-1-(cyclopropylamino)-1,2-dioxohexan-3-yl)octahydrocyclopenta[c]pyrrole-1-carboxamide[I-11]

The title compound was prepared according to the steps and intermediatesas described below.

Step 3a: Intermediate 3a

To a stirring solution of 372 mg of pyrazine-2-carboxylic acid (3 mmol),623 mg of (S)-methyl 2-amino-2-cyclohexylacetate (3 mmol), and 2.5 mL ofN,N-diisopropyl ethylamine in 25 mL of anhydrous dichloromethane, wasadded 760 mg of 1,4-dimethyl-2-chloroimidazolium hydrochloride (4.5mmol) in three portions. The resulting mixture was stirred at RT foradditional 30 min, then concentrated under reduced pressure. The residuewas re-dissolved into 80 mL of ethyl acetate, and washed with aqueoussodium bicarbonate, brine, and dried over anhydrous sodium sulfate.After concentration, the crude product was purified by flash columnchromatography, eluting with heptane/ethyl acetate (v/v 2/1), giving 897mg of yellowish oil as desired ester.

To a stirring solution of the ester obtained above in 4 mL of MeOH and 4mL of THF, was added 6 mL of 2.0 N LiOH aqueous solution. The mixturewas stirred at RT overnight, then 12 mL of 1.0 N aqueous HCl added. Thereaction mixture was extracted with dichloromethane 50 mL×2, and driedover anhydrous MgSO₄. After filtration, the filtrate was concentrated togive 700 mg of the title compound.

¹HNMR (CDCl₃, 400 MHz) δ 9.40 (d, 1H, J=1.4 Hz), 8.78 (d, 1H, J=2.2 Hz),8.58 (d, 1H, J=1.0 Hz), 8.25 (d, 1H, J=8.4 Hz), 4.78 (dd, 1H, J=4.8, 8.4Hz), 2.05 (m, 1H), 1.08 (m, 4H), 1.78 (m, 1H), 1.10-1.35 (m, 5H).

Step 3b: Intermediate 3b

To a stirring mixture of 263 mg of the product from step 3a (1 mmol),254 mg of (S)-methyl 2-amino-3-(tert-butoxycarbonylamino)propanoatehydrochloride (1 mmol), 1 mL of N,N-diisopropyl ethylamine in 10 mL ofacetonitrile, was added 450 mg of HATU (1.2 mmol). The reaction mixturewas stirred at RT overnight, then suspended in 60 mL of EtOAc and 15 mLof saturated NaHCO₃. The organic layer was separated and washed with 10mL of 1 N HCl, 15 mL of brine, dried over Na₂SO₄. After concentration,the residue was purified by flash column chromatography on silica gel,giving 463 mg of desired ester.

To a stirring solution of 463 mg of ester in 10 mL of THF and 10 mL ofMeOH, was added 10 mL of 1.0 N LiOH aqueous solution. The reactionmixture was stirred at RT for 30 min, LC-MS showed completion of esterhydrolysis. The solvent was removed under reduced pressure, and theresidue was extracted with 80 mL of dichloromethane. The organic layerwas washed with 20 mL of brine and dried with MgSO4. Afterconcentration, the title compound was obtained as white solid inquantitive yield.

LC-MS: 448.2 (ES−)

Step 3c: Intermediate 3c

To a mixture of 255 mg of(1S,3aR,6aS)-2-(tert-butoxycarbonyl)octahydrocyclopenta[c]pyrrole-1-carboxylicacid (1 mmol), 186 mg of (S)-3-amino-N-cyclopropyl-2-hydroxyhexanamide(1 mmol), and 1.0 mL of N,N-diisopropyl ethylamine in 10 mL ofacetonitrile, was added 400 mg of HATU (1.05 mmol). After 16 hr, themixture was diluted with 50 mL of EtOAc, and washed with aqueous NaHCO₃15 mL, 1 N aqueous HCl 10 mL, brine 10 mL, and dried over Na₂SO₄. Afterconcentration, the residue was purified by flash column chromatographyon silica gel (eluent 25% heptanes in EtOAc), giving 410 mg of desiredamide as white solid.

The Boc group of the amide obtained above was removed by stirring in 6mL of 4.0 M HCl in dioxane for 30 min. The solvent was then evaporatedunder reduced pressure, and the residue was dried in vacuum giving 350mg of white solid as desired product.

LC-MS: 324.2 (ES+)

Step 3d: Intermediate 3d

To a mixture of 274 mg of the product from step 3b (0.61 mmol), 220 mgof the product from step 3c (0.61 mmol), and 1.5 mL of N,N-diisopropylethylamine in 8 mL of acetonitrile, was added 300 mg of HATU (0.8 mmol).After 1 hr, the mixture was diluted with 100 mL of EtOAc, and washedwith aqueous NaHCO₃ 15 mL, 1 N aqueous HCl 10 mL, brine 10 mL, and driedover Na₂SO₄. After concentration, the residue was purified by flashcolumn chromatography on silica gel (eluent 5% MeOH in EtOAc), giving440 mg of desired amide as yellowish syrup (95%).

LC-MS: 753.4 (ES−)

Step 3e: Intermediate 3e

The title compound (217 mg, 53%) was prepared from the product of step3d following the procedures described in step 1e and 1f.

LC-MS: 709.3 (ES+), 707.3 (ES−)

Step 3f:(1S,3aR,6aS)-2-((S)-3-acrylamido-2-((S)-2-cyclohexyl-2-(pyrazine-2-carboxamido)acetamido)propanoyl)-N-((S)-1-(cyclopropylamino)-1,2-dioxohexan-3-yl)octahydrocyclopenta[c]pyrrole-1-carboxamide

To a stirring mixture of 217 mg of the product of step 3e (0.306 mmol)in 15 mL of dichloromethane, was added 390 mg of Dess-martin reagent(0.92 mmol). After 1 hr, LC-MS showed complete conversion. 15mL ofsaturated NaHCO₃ was added, and the stirring was continued for another 1hr. The organic layer was separated, and the aqueous layer was extractedone more time with 30 mL of dichloromethane. The combined organic layerwas washed with 20 mL of brine, and dried over MgSO₄. After filtrationand concentration, the residue was purified by Pre-HPLC using 0.1% TFAas modifier in water and acetonitrile, giving 45 mg of the titlecompound.

LC-MS: 707.3 (ES+), 705.3 (ES−)

In similar fashion starting from the product of step 3d (Intermediate3d), following the procedures described in step 3e and 3f (the de-boc,coupling and oxidation sequences), by using vinylsulfonylchloride inplace of acrylic acid and HATU, the following compound can be prepared:

Following the procedures described in example 3, using(S)-4-(tert-butoxycarbonylamino)-2-(3-tert-butylureido)butanoic acid tocouple with intermediate 3a in step 3b, the following compound can beprepared:

In similar fashion, using(S)-4-(tert-butoxycarbonylamino)-2-(3-tert-butylureido)butanoic acid instep step 3b, and using vinylsulfonylchloride in step 3e following theprocedures described in example 3, the following compound can beprepared:

Example 4

tert-butyl-(S)-1-((1S,3aR,6aS)-1-((S)-1-(cyclopropylamino)-1,2-dioxohexan-3-ylcarbamoyl)hexahydrocyclopenta[c]pyrrol-2(1H)-yl)-7-methyl-1,5-dioxooct-6-en-2-ylcarbamate

The title compound is prepared according to the steps and intermediatesas described below.

Step 4a: Intermediate 4a

The title compound is prepared by coupling intermediate 2a andintermediate 3c following the procedure described in step 2b in example2.

Step 4b: tert-butyl(S)-1-((1S,3aR,6aS)-1-((S)-1-(cyclopropylamino)-1,2-dioxohexan-3-ylcarbamoyl)hexahydrocyclopenta[c]pyrrol-2(1H)-yl)-7-methyl-1,5-dioxooct-6-en-2-ylcarbamate

The product from step 4a is oxidized to afford the title compoundfollowing the procedure described in step 2c in example 2.

Starting from the Intermediate 3c, by coupling with the appropriateintermediates made similarly as described in Step 2a, the followingcompounds are prepared:

Example 5

(1S,3aR,6aS)-2-((S)-2-((S)-2-cyclohexyl-2-(pyrazine-2-carboxamido)acetamido)-7-methyl-5-oxooct-6-enoyl)-N-((S)-1-(cyclopropylamino)-1,2-dioxohexan-3-yl)octahydrocyclopenta[c]pyrrole-1-carboxamide

The title compound is prepared according to the steps and intermediatesas described below.

Step 5a: Intermediate 5a

The product from step 4a is treated with 4N HCl to afford the titlecompound following the procedure described in step 1c in example 1.

Step 5b: Intermediate 5b

Intermediate 3a is coupled with Intermediate 5a with HATU following theprocedure described in step 3d in example 3 to produce the titlecompound.

Step 5c:(1S,3aR,6aS)-2-((S)-2-((S)-2-cyclohexyl-2-(pyrazine-2-carboxamido)acetamido)-7-methyl-5-oxooct-6-enoyl)-N-((S)-1-(cyclopropylamino)-1,2-dioxohexan-3-yl)octahydrocyclopenta[c]pyrrole-1-carboxamide

The product from step 5b is oxidized to afford the title compoundfollowing the procedure described in step 3f in example 3.

In similar fashion, the following compounds are prepared:

Example 6

(S)-3-((1S,3aR,6aS)-2-((S)-3-acrylamido-2-((S)-2-cyclohexyl-2-(pyrazine-2-carboxamido)acetamido)propanoyl)octahydrocyclopenta[c]pyrrole-1-carboxamido)-1-(cyclopropylamino)-1-oxohexan-2-yl2,2,2-trifluoroacetate, [I-23]

The title compound was prepared following steps 3a to 3e, by using(S)-3-amino-1-(cyclopropylamino)-1-oxohexan-2-yl 2,2,2-trifluoroacetatein step 3c in place of (S)-3-amino-N-cyclopropyl-2-hydroxyhexanamide.

LC-MS: 805.3 (ES+)

Example 7

Single Chain HCV Protease (wt) Peptide Expression and Purification

The single-chain proteolytic domain (NS4A₂₁₋₃₂-GSGS-NS₃₃₋₆₃₁) was clonedinto pET-14b (Novagen, Madison, Wis.) and transformed into DH10B cells(Invitrogen). The resulting plasmid was transferred into Escherichiacoli BL21 (Novagen) for protein expression and purification as describedpreviously (1, 2). Briefly, the cultures were grown at 37° C. in LBmedium containing 100 μg/mL of ampicillin until the optical density at600 nm (OD600) reached 1.0 and were induced by addition ofisopropyl-β-D-thiogalactopyranoside (IPTG) to 1 mM. After an additionalincubation at 18° C. for 20 h, bacteria were harvested by centrifugationat 6,000×g for 10 min and resuspended in a lysis buffer containing 50 mMNa₃PO₄, pH 8.0, 300 mM NaCl, 5 mM 2-mercaptoethanol, 10% glycerol, 0.5%Igepal CA630, and a protease inhibitor cocktail consisting of 1 mMphenylmethylsulfonyl fluoride, 0.5 μg/mL leupeptin, pepstatin A, and 2mM benzamidine. Cells were lysed by freezing and thawing, followed bysonication. Cell debris was removed by centrifugation at 12,000×g for 30min. The supernatant was further clarified by passing through a 0.45-μmfilter (Corning) and then loaded onto a HiTrap chelating column chargedwith NiSO₄ (Amersham Pharmacia Biotech). The bound protein was elutedwith an imidazole solution in a 100-to-500 mM linear gradient. Selectedfractions were run through Ni²⁺ column chromatography and were analyzedon a 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel. The purifiedprotein was resolved by electrophoresis in a 12% SDS-PAGE gel and thentransferred onto a nitrocellulose membrane. The protein was analyzed byWestern blot analysis using monoclonal antibodies against NS3. Proteinswere visualized by using a chemiluminescence kit (Roche) withhorseradish peroxidase-conjugated goat anti-mouse antibodies (Pierce) assecondary antibodies. The protein was aliquoted and stored at −80° C.

Example 8

Cloning and Expression of HCV Protease A156S, A156T, D168A, D168VDrug-Resistance Mutants and C159S Variant

The mutant DNA fragments of NS4A/NS3 were generated by PCR and clonedinto pET expression vector. After transformation into BL21 competentcells, the expression was induced with IPTG for 2 hours. The His-taggedfusion proteins were purified using affinity column followed by sizeexclusion chromatography.

Example 9

Assay buffer: 2% CHAPS, 50 mM Tris pH 7.5, 50% glycerol, 2 uM M-2235(Bachem) substrate. In a 50 ul reaction, add 49 ul assay buffer, 1 ul(1U) HCV serine protease (Bioenza). Incubate 20 minutes at roomtemperature. The plate was read at either 350/460 nm(excitation/emission) on a fluorescent micro-plate reader or monitoredat one-minute intervals to achieve the kinetic curve.

The enzyme tolerated 1% DMSO and 2% methanol. In the experiments oftesting compounds, the compounds in pure DMSO were diluted 10 times with20% methanol (10% DMSO and 20% methanol). This compound solution wasadded to the reaction (not exceeding 10% of the final reaction volume).The final concentration of the organic solvents was: 1% DMSO and 2%methanol.

Example 10

Additional Assay Protocols

Method A:

The compounds were assayed to evaluate the antiviral activity andcytotoxicity of compounds in vitro using HCV RNA replicons. This assayused the cell line ET (luc-ubi-neo/ET), which is a human Huh7 hepatomacell line that contains an HCV RNA replicon with a stable luciferase(Luc) reporter and three cell culture-adaptive mutations. The HCV RNAlevels were directly measured by viral specific TaqMan RT-PCR:

Forward primer: (SEQ ID NO: 63) ACGCAGAAAGCGTCTAGCCAT Reverse primer:(SEQ ID NO: 64) TACTCACCGGTTCCGCAGA Probe: (SEQ ID NO: 65)[6-FAM]-CCTGGAGGCTGCACGACACTCAT-[TAMRA]

The ET cell line was grown in Dulbecco's modified essential media(DMEM), 10% fetal bovine serum (FBS), 1% penicillin-streptomycin(pen-strep), 1% glutamine, 250 μg/mL G418 in a 5% CO₂ incubator at 37°C. All cell culture reagents were obtained from Mediatech (Manassas,Va.). Cells were trypsinized (1% trypsin:EDTA) and plated out at 5×10³cells/well in white 96-well assay plates (Costar) dedicated to cellnumber (cytotoxicity) or antiviral activity assessments. Drugs wereadded at six 3-fold concentrations each and the assay was run in DMEM,5% FBS, 1% pen-strep, 1% glutamine. Human interferon alpha-2b (PBLBiolabs, New Brunswick, N.J.) was included in each run as a positivecontrol compound. Cells were processed 72 hr post drug addition when thecells are still subconfluent. Antiviral activity was measured byanalyzing replicon-derived luciferase activity using the Steady-GloLuciferase Assay System (Promega, Madison, Wis.) according tomanufacturer's instruction. The number of cells in each well wasdetermined by CytoTox-1 reagent (Promega). Compound profile was derivedby calculating applicable EC₅₀ (effective concentration inhibiting virusreplication by 50%), EC₉₀ (effective concentration inhibiting virusreplication by 90%), IC₅₀ (concentration decreasing cell viability by50%) and SI₅₀ (selective index: EC₅₀/IC₅₀) values. IC₅₀ values forselected compounds are set forth in Table 5, below.

Method B: HCV Protease Assay Using FRET Methodology

A quantitative, fluorescence resonance energy transfer (FRET)-basedmethodology was employed to identify HCV NS3/4A protease inhibitors. Theassay employed a synthetic FRET peptide, derived from the HCV NS5A/5Bcleavage site, with the HCV protease to evaluate the activity ofcompounds against the protease by monitoring the cleavage activity ofthe complex. A synthetic peptide which encompasses the NS5A-5B junction(NH2-EDVVCCSMSYK-COOH) (SEQ ID NO: 78) was labeled with Dabcyl and Edansat N- and C-termini, respectively (Invitrogen, Carlsbad, Calif.).Fluorescence measurement was used to estimate the IC₅₀ value of the testcompound. The two fluorophores form a quenching pair and exhibit FRETwithin the intact peptide. Upon cleavage of the FRET peptide by HCVNS3/4A proteinase complex (10 ng/mL), the fluorescence is recovered andcan be continuously monitored at excitation/emission=340/490 nm.

Example 11

HCV Protease FRET Assay for Mutated NS3/4A 1b Enzymes

The following protocol was used to generate IC₅₀ values as depicted forcompounds in Tables 4b and 5. The protocol is a modified FRET-basedassay (v_(—)02) from In Vitro Resistance Studies of HCV Serine ProteaseInhibitors, 2004, JBC, vol. 279, No. 17, pp 17508-17514. Inherentpotency of compounds was assessed against A156S, A156T, D168A, and D168Vmutants of the HCV NS3/4A 1b protease enzyme as follows:

10× stocks of NS3/4A protease enzyme from Bioenza (Mountain View,Calif.) and 1.13×5-FAM/QXL™520 FRET peptide substrate from Anaspec (SanJose, Calif.) were prepared in 50 mM HEPES, pH 7.8, 100 mM NaCl, 5 mMDTT and 20% glycerol. 5 μL of each enzyme were pre-incubated in aCorning (#3573) 384-well, black, non-treated microtiter plate (Corning,N.Y.) for 30 min at 25° C. with a 0.5 μL volume of 50% DMSO and seriallydiluted compounds prepared in 50% DMSO. Protease reactions were startedwith the addition of 45 μL of the FRET substrate and monitored for 120minutes at λ_(ex)487/λ_(em)514 through Quad⁴ monochromoters in aSynergy⁴ plate reader from BioTek (Winooski, Vt.). At the conclusion ofeach assay, progress curves from each well were examined for linearreaction kinetics and fit statistics (R², absolute sum of squares).Initial velocity (0 minutes to 30+ minutes) from each reaction wasdetermined from the slope of a plot of relative fluorescence units vstime (minutes) and then plotted against inhibitor concentration toestimate IC₅₀ from log [Inhibitor] vs Response, Variable Slope model inGraphPad Prism from GraphPad Software (San Diego, Calif.). IC₅₀ valuesfor selected compounds are set forth in Table 5, below.

TABLE 5 Enzymatic Data for Exemplary Compounds (IC₅₀) Compound testedEnzyme/Assay IC₅₀ (nM)¹ (I-1) WT 290 HCV D168A 312 HCV R155K 928 HCVC159S 1090 (I-6) WT 53 HCV D168A 25  (I-11) WT 232 D168A 98 ¹Valuesgreater than one were rounded to the nearest whole number.

Example 12

HCV Protease FRET Assay for WT and Mutated NS3/4A 1b Enzymes (IC₅₀ _(—)_(APP)).

The following protocol was used to generate “apparent” IC₅₀ (IC₅₀ _(—)_(APP)) values as depicted in Table 6, below. Without wishing to bebound by any particular theory, it is believed that IC₅₀ _(—) _(APP),constrasted with IC₅₀ values, may provide a more useful indication oftime-dependent inhibition, and are thus more representative of bindingaffinity. The protocol is a modified FRET-based assay (v_(—)03)developed to evaluate compound potency, rank-order and resistanceprofiles against wild type and C159S, A156S, A156T, D168A, D168V, R155Kmutants of the HCV NS3/4A 1b protease enzyme as follows: 10× stocks ofNS3/4A protease enzyme from Bioenza (Mountain View, Calif.) and1.13×5-FAM/QXL™520 FRET peptide substrate from Anaspec (San Jose,Calif.) were prepared in 50 mM Tris-HCl, pH 7.5, 5 mM DTT, 2% CHAPS and20% glycerol. 5 μL of each enzyme were added to Corning (#3575)384-well, black, microtiter plates (Corning, N.Y.) after spotting a 0.5μL volume of 50% DMSO and serially diluted compounds prepared in 50%DMSO. Protease reactions were immediately started after enzyme additionwith the addition of 45 μL of the FRET substrate and monitored for 60-90minutes at λ_(ex)485/λ_(cm)520 in a Synergy⁴ plate reader from BioTek(Winooski, Vt.). At the conclusion of each assay, progress curves fromeach well were examined for linear reaction kinetics and fit statistics(R², 95% confidence intervals, absolute sum of squares). Initialvelocity (0 minutes to 15+ minutes) from each reaction was determinedfrom the slope of a plot of relative fluorescence units vs time(minutes) and then plotted against inhibitor concentration as a percentof the no inhibitor and no enzyme controls to estimate apparent IC₅₀from log [Inhibitor] vs Response, Variable Slope model in GraphPad Prismfrom GraphPad Software (San Diego, Calif.).

TABLE 6 Enzymatic Data for Exemplary Compounds Compound testedEnzyme/Assay IC₅₀ _(—) _(APP) (nM)¹ (I-1)  WT 1330 HCV A156S >3000 HCVD168A 2550 HCV R155K >3000 HCV C159S >3000 (I-6)  WT 456 HCV A156S >3000HCV D168A 321 HCV R155K 1050 HCV C159S 2070 (I-11) WT 1740 HCVA156S >3000 HCV D168A 1150 HCV R155K >3000 HCV C159S >3000 (I-23)WT >111 HCV A156S 412 HCV A156T 985 HCV D168A 508 HCV D168V 463 ¹Valuesgreater than one were rounded to the nearest whole number.

Example 13

Mass spectrometric analysis of HCV wild type or HCV variant C159S in thepresence of test compound is performed. 100 pmols of HCV wild type(Bioenza Calif.) is incubated with test compound for 1 hr and 3 hrs at10-fold access of test compound to protein. 1 ul aliquots of the samples(total volume of 4.24 ul) are diluted with 10 ul of 0.1% TFA prior tomicro C4 ZipTipping directly onto the MALDI target using Sinapinic acidas the desorption matrix (10 mg/mL in 0.1% TFA:Acetonitrile 50:50).Analyses are performed on a Shimadzu Biotech Axima TOF² (ShimadzuInstruments) matrix-assisted-laser desorption/ionization Time-of-Flight(MALDI-TOF) mass spectrometer. The same procedure is carried out on 100pmols of HCV C159S mutant of HCV protease for 3 hrs at 10-fold excess oftest compound to protein.

Example 14

Modification of Cys159 of Wild-Type HCV Protease Using a Tryptic DigestStrategy

HCV is incubated with test compound for 3 hrs prior to trypticdigestion. Iodoacetamide is used as the alkylating agent after compoundincubation. For tryptic digests a 2 ul aliquot (0.06 ug/ul) is dilutedwith 10 ul of 0.1% TFA prior to micro C18 Zip Tipping directly onto theMALDI target using alpha cyano-4-hydroxy cinnamic acid as the matrix (5mg/mL in 0.1% TFA:Acetonitrile 50:50).

For tryptic digests the instrument is set in Reflectron mode with apulsed extraction setting of 1800. Calibration is done using the LaserBiolabs Pep Mix standard (1046.54, 1296.69, 1672.92, 2093.09, 2465.20).For CID/PSD analysis the peptide is selected using cursors to set iongate timing and fragmentation occurred at a laser power about 20% higherand He is used as the collision gas for CID. Calibration for fragmentsis done using the P14R fragmentation calibration for the Curved fieldReflectron.

Example 15

As depicted in FIGS. 1 and 3, mass spectrometric analysis of HCV wildtype in the presence of compounds I-1 and telaprevir was performed usingthe following protocol: HCV NS3/4A wild type (wt) was incubated for 1 hrat a 10× fold access of test compound to protein. 2 ul aliquots of thesamples were diluted with 10 ul of 0.1% TFA prior to micro C4 ZipTippingdirectly onto the MALDI target using Sinapinic acid as the desorptionmatrix (10 mg/ml in 0.1% TFA:Acetonitrile 50:50). For intact proteinmass measurement the instrument was set in linear mode using a pulsedextraction setting of 24,500 and apomyoglobin as the standard tocalibrate the instrument.

As depicted in FIG. 1 (upper panel), compared to the protein with nocompound, the protein incubated with compound I-1 has reactedsignificantly to produce a new species at MW 25,004 Da, which isapproximately 537 Da heavier and consistent with the mass of compoundI-1 at 547 Da. The lower panel shows no modification of the C159S mutant(see Example 13).

As depicted in FIG. 3, after 1 hour incubation there was no reactionbetween the protein and telaprevir.

Example 16

As depicted in FIG. 2, mass spectrometric analysis of HCV wild type inthe presence of compound I-11 was performed using the followingprotocol: HCV NS3/4A wild type (wt) was incubated for 3 hr at a 10× foldaccess of test compound to protein. 2 ul aliquots of the samples werediluted with 10 ul of 0.1% TFA prior to micro C18 ZipTipping directlyonto the MALDI target using Sinapinic acid as the desorption matrix (10mg/ml in 0.1% TFA:Acetonitrile 20:80). For intact protein massmeasurement the instrument was set in linear mode using a pulsedextraction setting of 24,500 and apomyoglobin as the standard tocalibrate the instrument.

As depicted in FIG. 2, compared to the protein with no compound, theprotein incubated with compound I-11 has reacted significantly toproduce a new species at MW 25,155 Da, which is approximately 703 Daheavier and consistent with the mass of compound I-11 at 707 Da.

Example 17

Cell Culture

Huh-luc/neo-ET, Huh7-Lunet were obtained from ReBLikon Gmbh (Heidelberg,Germany). Cells were grown in Dulbecco modified Eagle medium (DMEM;Invitrogen) supplemented with 2 mM L-glutamine, nonessential aminoacids, 100 U of penicillin/ml, 100 μg of streptomycin/mL, and 10% fetalbovine serum. G418 (Geneticin; Invitrogen) was added at a finalconcentration of 400 ug//mL. Huh7-Lunet were grown in the absence ofG418.

Example 18

Mutant Constructs

Constructs containing clinically relevant mutations were generated byperforming site-directed mutagenesis on thepFK-I389-luc-ubi-neo-NS3-3′ET plasmid (ReBLikon Gmbh (Heidelberg,Germany)). using the QuickChange II Site-Directed Mutagenesis Kit(Stratagene, La Jolla, Calif.) according to manufacturer's directionsand with the primers described in Table 7, below.

TABLE 7 Primer sequence used to establish Mutant Replicon cell lines.NS3-A156S-F GCTGTGGGCATCTTTCGGTCTGCCGT SEQ ID NO: 66 GTGCACCCGAGGGNS3-A156S-R CCCTCGGGTGCACACGGCAGACCGAA SEQ ID NO: 67 AGATGCCCACAGCNS3-A156T-F GCTGTGGGCATCTTTCGGACTGCCGT SEQ ID NO: 68 GTGCACCCGAGGGNS3-A156T-R CCCTCGGGTGCACACGGCAGTCCGAA SEQ ID NO: 69 AGATGCCCACAGCNS3-D168A-F GGGGTTGCGAAGGCGGTGGCCTTTGT SEQ ID NO: 70 ACCCGTCGAGTCTNS3-D168A-R AGACTCGACGGGTACAAAGGCCACCG SEQ ID NO: 71 CCTTCGCAACCCCNS3-D168V-F GGGGTTGCGAAGGCGGTGGTCTTTGT SEQ ID NO: 72 ACCCGTCGAGTCTNS3-D168V-R AGACTCGACGGGTACAAAGACCACCG SEQ ID NO: 73 CCTTCGCAACCCCNS3-C159S-F ATCTTTCGGGCTGCCGTGAGCACCCG SEQ ID NO: 74 AGGGGTTGCGAAGNS3-C159S-R CTTCGCAACCCCTCGGGTGCTCACGG SEQ ID NO: 75 CAGCCCGAAAGATNS3-R155K-F CACGCTGTGGGCATCTTTAAGGCTGC SEQ ID NO: 76 CGTGTGCACCCGANS3-R155K-R TCGGGTGCACACGGCAGCCTTAAAGA SEQ ID NO: 77 TGCCCACAGCGTG

Example 19

In vitro Transcription

In vitro transcripts of HCV positive strands were generated by using theprotocol described by Lohmann V et al., J. Virol., 77:3007-3019, 2003.For transcription of positive-strand HCV RNAs, plasmid DNA (pFK 1341PI-Luc/NS3-3′/ET, obtained from ReBLikon Gmbh (Heidelberg, Germany)),was digested with Ase1 followed by Sca1. After restriction digest, DNAwas extracted with phenol and chloroform, precipitated with ethanol, anddissolved in RNase-free water. In vitro transcription reactionscontained 80 mM HEPES (pH 7.5), 12 mM MgCl₂, 2 mM spermidine, 40 mMdithiothreitol, a 3.125 mM concentration of each nucleosidetriphosphate, 1 U of RNasin. 5 ug of restricted plasmid DNA and 80 U ofT7 RNA polymerase (Promega) was used. After 2 h at 37° C., an additional40 U of T7 polymerase was added, and the reaction was incubated foranother 2 h. Transcription was terminated by the addition of 1 U ofRNase-free DNase (Promega) per ug of plasmid DNA, followed by incubationfor 30 min at 37° C. After extraction with acidic phenol and chloroform,RNA was precipitated with isopropanol and dissolved in RNase-free water.The concentration was determined by measurement of the optical densityat 260 nm (OD260), and RNA integrity was checked by denaturing agarosegel electrophoresis.

Example 20

Transfection of HCV Full Length Genome and Selection of Stable CellLines

7×10⁴ Huh7-Lunet cells were seeded over night in a 12 well plate, thenext day 1 ug of RNA/well was transfected using Mirus Tx (Madison, Wis.)kit. Transfection was performed according to manufacturer'sinstructions, and 24 hours after transfection cells were eithersubjected to Luciferase assay or subjected to G418 (400 ug/ml) selectionin order to establish stable cell lines.

Example 21

Luciferase Assay

The compounds are assayed to evaluate the antiviral activity andcytotoxicity of compounds using replicon-derived luciferase activity.This assay uses the cell line ET (luc-ubi-neo/ET), which is a human Huh7hepatoma cell line that contains an HCV RNA replicon with a stableluciferase (Luc) reporter and cell culture-adaptive mutations. The ETcell line is grown in a 5% CO₂ incubator at 37° C. in Dulbecco'smodified essential media (DMEM) supplemented with 2 mM L-glutamine,nonessential amino acids, 100 U of penicillin/ml, 100 μg ofstreptomycin/mL, and 10% fetal bovine serum. G418 (Geneticin;Invitrogen) is added at a final concentration of 400 ug//mL.

All cell culture reagents are obtained from Invitrogen (Carlsbad). Cellsare trypsinized (1% trypsin:EDTA) and plated out at 5×10³ cells/well inwhite 96-well assay plates (Costar) dedicated to cell number(cytotoxicity) or antiviral activity assessments. Test compounds areadded at six 3-fold concentrations each and the assay is run in DMEM, 5%FBS, 1% pen-strep, 1% glutamine, 1% non essential amino acid. Humaninterferon alpha-2b (PBL Biolabs, New Brunswick, N.J.) is included ineach run as a positive control compound. Cells are processed 72 hr posttest compound addition when the cells are still subconfluent. Antiviralactivity is measured by analyzing replicon-derived luciferase activityusing the Steady-Glo Luciferase Assay System (Promega, Madison, Wis.)according to manufacturer's instruction. The number of cells in eachwell is determined by Cell Titer Blue Assay (Promega). Compound profileis derived by calculating applicable EC₅₀ (effective concentrationinhibiting virus replication by 50%), EC₉₀ (effective concentrationinhibiting virus replication by 90%), IC₅₀ (concentration decreasingcell viability by 50%) and SI₅₀ (selective index: EC₅₀/IC₅₀) values.

While we have described a number of embodiments of this invention, it isapparent that our basic examples may be altered to provide otherembodiments that utilize the compounds and methods of this invention.Therefore, it will be appreciated that the scope of this invention is tobe defined by the appended claims rather than by the specificembodiments that have been represented by way of example.

1. A compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein: one of R^(a) andR^(b) is hydrogen and the other is —OH or —OC(O)R′, or R^(a) and R^(b)are taken together to form an oxo group; R′ is an optionally substitutedgroup selected from C₁₋₆ aliphatic, C₃₋₇ cycloalkyl, 6-10 membered aryl,5-10 membered heteroaryl having 1-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur, or 4-7 membered heterocyclyl having1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur;R^(x) and R^(y) are taken together to form an optionally substitutedC₃₋₇ membered ring having 0-2 heteroatoms independently selected fromnitrogen, oxygen, or sulfur; R¹ is an optionally substituted groupselected from C₁₋₆ aliphatic or C₃₋₇ cycloalkyl(C₁₋₃ alkyl); R² ishydrogen or an optionally substituted group selected from C₁₋₆ aliphaticor C₃₋₇ cycloalkyl; R³ is a warhead group —(CH₂)_(n)-L-Y, wherein n isan integer from 0 to 5, inclusive, and -L-Y is selected from thefollowing (a), (b), (c), (h), (i), (k), (l), (m), (n), (o), and (p)combinations of -L- and —Y: (a) L is a bivalent C₂₋₈ straight orbranched, hydrocarbon chain wherein L has at least one double bond andone or two additional methylene units of L are optionally andindependently replaced by —NRC(O)—, —C(O)NR—, —N(R)SO₂—, —SO₂N(R)—, —S—,—S(O)—, —SO₂—, —OC(O)—, —C(O)O—, cyclopropylene, —O—, —N(R)—, or —C(O)—;and Y is hydrogen or C₁₋₆ aliphatic optionally substituted with oxo,halogen, NO₂, or CN; or (b) L is a bivalent C₂₋₈ straight or branched,hydrocarbon chain wherein L has at least one double bond and at leastone methylene unit of L is replaced by —C(O)—, —NRC(O)—, —C(O)NR—,—N(R)SO₂—, —SO₂N(R)—, —S—, —S(O)—, —SO₂—, —OC(O)—, or —C(O)O—, and oneor two additional methylene units of L are optionally and independentlyreplaced by cyclopropylene, —O—, —N(R)—, or —C(O)—; and Y is hydrogen orC₁₋₆ aliphatic optionally substituted with oxo, halogen, NO₂, or CN; or(c) L is a bivalent C₂₋₈ straight or branched, hydrocarbon chain whereinL has at least one double bond and at least one methylene unit of L isreplaced by —C(O)—, and one or two additional methylene units of L areoptionally and independently replaced by cyclopropylene, —O—, —N(R)—, or—C(O)—; and Y is hydrogen or C₁₋₆ aliphatic optionally substituted withoxo, halogen, NO₂, or CN; or (h) L is a bivalent C₂₋₈ straight orbranched, hydrocarbon chain wherein L has at least one alkylidenyldouble bond and at least one methylene unit of L is replaced by —C(O)—,—NRC(O)—, —C(O)NR—, —N(R)SO₂—, —SO₂N(R)—, —S—, —S(O)—, —SO₂—, —OC(O)—,or —C(O)O—, and one or two additional methylene units of L areoptionally and independently replaced by cyclopropylene, —O—, —N(R)—, or—C(O)—; and Y is hydrogen or C₁₋₆ aliphatic optionally substituted withoxo, halogen, NO₂, or CN; or (i) L is a bivalent C₂₋₈ straight orbranched, hydrocarbon chain wherein L has at least one triple bond andone or two additional methylene units of L are optionally andindependently replaced by —NRC(O)—, —C(O)NR—, —N(R)SO₂—, —SO₂N(R)—, —S—,—S(O)—, —SO₂—, —OC(O)—, or —C(O)O—, and Y is hydrogen or C₁₋₆ aliphaticoptionally substituted with oxo, halogen, NO₂, or CN; or (k) L is abivalent C₂₋₈ straight or branched, hydrocarbon chain wherein onemethylene unit of L is replaced by cyclopropylene and one or twoadditional methylene units of L are independently replaced by —NRC(O)—,—C(O)NR—, —N(R)SO₂—, —SO₂N(R)—, —S—, —S(O)—, —SO₂—, —OC(O)—, or —C(O)O—;and Y is C₁₋₆ aliphatic substituted with oxo, halogen, NO₂, or CN; or(l) L is a covalent bond and Y is selected from: (i) C₁₋₆ alkylsubstituted with oxo, halogen, NO₂, or CN; (ii) C₂₋₆ alkenyl optionallysubstituted with oxo, halogen, NO₂, or CN; or (iii) C₂₋₆ alkynyloptionally substituted with oxo, halogen, NO₂, or CN; or (vii) asaturated 3-6 membered carbocyclic ring, wherein said ring issubstituted with 1-4 R^(e) groups; or (ix) a partially unsaturated 3-6membered carbocyclic ring, wherein said ring is substituted with 1-4R^(e) groups; or (xvii) an 8-10 membered bicyclic, saturated orpartially unsaturated ring, wherein said ring is substituted with 1-4R^(e) groups; (m) L is —C(O)—and Y is selected from: (i) C₁₋₆ alkylsubstituted with oxo, halogen, NO₂, or CN; or (ii) C₂₋₆ alkenyloptionally substituted with oxo, halogen, NO₂, or CN; or (iii) C₂₋₆alkynyl optionally substituted with oxo, halogen, NO₂, or CN; or (vii) asaturated 3-6 membered carbocyclic ring, wherein said ring issubstituted with 1-4 R^(e) groups; or (ix) a partially unsaturated 3-6membered carbocyclic ring, wherein said ring is substituted with 1-4R^(e) groups; or (xvii) an 8-10 membered bicyclic, saturated orpartially unsaturated ring, wherein said ring is substituted with 1-4R^(e) groups; (n) L is —N(R)C(O)—and Y is selected from: (i) C₁₋₆ alkylsubstituted with oxo, halogen, NO₂, or CN; or (ii) C₂₋₆ alkenyloptionally substituted with oxo, halogen, NO₂, or CN; or (iii) C₂₋₆alkynyl optionally substituted with oxo, halogen, NO₂, or CN; or (vii) asaturated 3-6 membered carbocyclic ring, wherein said ring issubstituted with 1-4 R^(e) groups; or (ix) a partially unsaturated 3-6membered carbocyclic ring, wherein said ring is substituted with 1-4R^(e) groups; or (xvii) an 8-10 membered bicyclic, saturated orpartially unsaturated ring, wherein said ring is substituted with 1-4R^(e) groups; (o) L is a bivalent C₁₋₈ saturated or unsaturated,straight or branched, hydrocarbon chain; and Y is selected from: (i)C₁₋₆ alkyl substituted with oxo, halogen, NO₂, or CN; (ii) C₂₋₆ alkenyloptionally substituted with oxo, halogen, NO₂, or CN; or (iii) C₂₋₆alkynyl optionally substituted with oxo, halogen, NO₂, or CN; or (vii) asaturated 3-6 membered carbocyclic ring, wherein said ring issubstituted with 1-4 R^(e) groups; or (ix) a partially unsaturated 3-6membered carbocyclic ring, wherein said ring is substituted with 1-4R^(e) groups; or (xvii) an 8-10 membered bicyclic, saturated orpartially unsaturated ring, wherein said ring is substituted with 1-4R^(e) groups; (p) L is a covalent bond, —CH₂—, —NH—, —C(O)—, —CH₂NH—,—NHCH₂—, —NHC(O)—, —NHC(O)CH₂OC(O)—, —CH₂NHC(O)—, —NHSO₂—, —NHSO₂CH₂—,—NHC(O)CH₂OC(O)—, or —SO₂NH—; and Y is selected from: (i) C₁₋₆ alkylsubstituted with oxo, halogen, NO₂, or CN; or (ii) C₂₋₆ alkenyloptionally substituted with oxo, halogen, NO₂, or CN; or (iii) C₂₋₆alkynyl optionally substituted with oxo, halogen, NO₂, or CN; or (vii) asaturated 3-6 membered carbocyclic ring, wherein said ring issubstituted with 1-4 R^(e) groups; or (ix) a partially unsaturated 3-6membered carbocyclic ring, wherein said ring is substituted with 1-4R^(e) groups; or (xvii) an 8-10 membered bicyclic, saturated orpartially unsaturated ring, wherein said ring is substituted with 1-4R^(e) groups; each R^(e) is independently selected from -Q-Z, oxo, NO₂,halogen, CN, a suitable leaving group selected from alkoxy,sulphonyloxy, optionally substituted alkylsulphonyloxy, optionallysubstituted alkenylsulfonyloxy, optionally substituted arylsulfonyloxy,acyl, diazonium, or C₁₋₆ aliphatic optionally substituted with oxo,halogen, NO₂, or CN, wherein: Q is a covalent bond or a bivalent C₁₋₆saturated or unsaturated, straight or branched, hydrocarbon chain,wherein one or two methylene units of Q are independently replaced by—N(R)—, —S—, —O—, —C(O)—, —OC(O)—, —C(O)O—, —SO—, or —SO₂—, —N(R)C(O)—,—C(O)N(R)—, —N(R)SO₂—, or —SO₂N(R)—; and each Z is independentlyhydrogen or C₁₋₆ aliphatic optionally substituted with oxo, halogen,NO₂, or CN; R⁴ is —NHC(O)NHR⁵, —NHC(O)OR⁶, or

R⁵ is an optionally substituted group selected from C₁₋₆ aliphatic,bridged bicyclic, 6-10 membered aryl, 5-10 membered heteroaryl having1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur,or 4-7 membered heterocyclyl having 1-2heteroatoms independentlyselected from nitrogen, oxygen, or sulfur; R⁶ is an optionallysubstituted group selected from C₁₋₆ aliphatic, bridged bicyclic, 6-10membered aryl, 5-10 membered heteroaryl having 1-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or 4-7 memberedheterocyclyl having 1-2 heteroatoms independently selected fromnitrogen, oxygen, or sulfur; R⁷ is an optionally substituted groupselected from C₁₋₆ aliphatic, bridged bicyclic, 6-10 membered aryl, 5-10membered heteroaryl having 1-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, or 4-7 membered heterocyclyl having 1-2heteroatoms independently selected from nitrogen, oxygen, or sulfur; andR is hydrogen or an optionally substituted group selected from C₁₋₆aliphatic, C₃₋₇ cycloalkyl, 6-10 membered aryl, 5-10 membered heteroarylhaving 1-4 heteroatoms independently selected from nitrogen, oxygen, orsulfur, or 4-7 membered heterocyclyl having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur.
 2. The compoundaccording to claim 1, wherein the compound is of formula II-a or II-b:

or a pharmaceutically acceptable salt thereof.
 3. The compound accordingto claim 2, wherein the compound is of formula III-a or III-b:

or a pharmaceutically acceptable salt thereof, wherein: R² is anoptionally substituted group selected from C₁₋₆ aliphatic or C₃₋₇cycloalkyl.
 4. The compound according to claim 2, wherein the compoundis of formula IV-a or IV-b:

or a pharmaceutically acceptable salt thereof.
 5. The compound accordingto claim 2, wherein the compound is of formula V-a or V-b:

or a pharmaceutically acceptable salt thereof.
 6. The compound accordingto claim 4, wherein the compound is of formula IV-a-1 or IV-a-2:


7. The compound according to claim 4, wherein the compound is of formulaIV-b-1 or IV-b-2:


8. The compound according to claim 5, wherein the compound is of formulaV-a-1 or V-a-2:


9. The compound according to claim 5, wherein the compound is of formulaV-b-1 or V-b-2:


10. A compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein: one of R^(a) andR^(b) is hydrogen and the other is —OH or —OC(O)R′, or R^(a) and R^(b)are taken together to form an oxo group; R′ is an optionally substitutedgroup selected from C₁₋₆ aliphatic, C₃₋₇ cycloalkyl, 6-10 membered aryl,5-10 membered heteroaryl having 1-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur, or 4-7 membered heterocyclyl having1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur;R^(x) and R^(y) are taken together to form an optionally substitutedC₃₋₇ membered ring having 0-2 heteroatoms independently selected fromnitrogen, oxygen, or sulfur; R¹ is an optionally substituted groupselected from C₁₋₆ aliphatic or C₃₋₇ cycloalkyl(C₁₋₃ alkyl); R² ishydrogen or an optionally substituted group selected from C₁₋₆ aliphaticor C₃₋₇ cycloalkyl; R³ is a warhead —(CH₂)_(n)-L-Y, wherein: n is aninteger from 0 to 5, inclusive; L is a bivalent C₂₋₈ straight orbranched, hydrocarbon chain wherein L has at least one double bond andone or two additional methylene units of L are optionally andindependently replaced by —NRC(O)—, —C(O)NR—, —N(R)SO₂—, —SO₂N(R)—, —S—,—S(O)—, —SO₂—, —OC(O)—, —C(O)O—, cyclopropylene, —O—, —N(R)—, or —C(O)—;Y is hydrogen, C₁₋₆ aliphatic optionally substituted with oxo, halogen,NO₂, or CN, or a 3-10 membered monocyclic or bicyclic, saturated,partially unsaturated, or aryl ring having 0-3 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur, and wherein said ring issubstituted with 1-4 R^(e) groups; and each R^(e) is independentlyselected from -Q-Z, oxo, NO₂, halogen, CN, a suitable leaving groupselected from alkoxy, sulphonyloxy, optionally substitutedalkylsulphonyloxy, optionally substituted alkenylsulfonyloxy, optionallysubstituted arylsulfonyloxy, acyl, diazonium, or C₁₋₆ aliphaticoptionally substituted with oxo, halogen, NO₂, or CN, wherein: Q is acovalent bond or a bivalent C₁₋₆ saturated or unsaturated, straight orbranched, hydrocarbon chain, wherein one or two methylene units of Q areindependently replaced by —N(R)—, —S—, —O—, —C(O)—, —OC(O)—, —C(O)O—,—SO—, or —SO₂—, —N(R)C(O)—, —C(O)N(R)—, —N(R)SO₂—, or —SO₂N(R)—; and Zis hydrogen or C₁₋₆ aliphatic optionally substituted with oxo, halogen,NO₂, or CN; R⁴ is —NHC(O)NHR⁵, —NHC(O)OR⁶, or

R⁵ is an optionally substituted group selected from C₁₋₆ aliphatic,bridged bicyclic, 6-10 membered aryl, 5-10 membered heteroaryl having1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur,or 4-7 membered heterocyclyl having 1-2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur; R⁶ is an optionallysubstituted group selected from C₁₋₆ aliphatic, bridged bicyclic, 6-10membered aryl, 5-10 membered heteroaryl having 1-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or 4-7 memberedheterocyclyl having 1-2 heteroatoms independently selected fromnitrogen, oxygen, or sulfur; R⁷ is an optionally substituted groupselected from C₁₋₆ aliphatic, bridged bicyclic, 6-10 membered aryl, 5-10membered heteroaryl having 1-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, or 4-7 membered heterocyclyl having 1-2heteroatoms independently selected from nitrogen, oxygen, or sulfur; andR is hydrogen or an optionally substituted group selected from C₁₋₆aliphatic, C₃₋₇ cycloalkyl, 6-10 membered aryl, 5-10 membered heteroarylhaving 1-4 heteroatoms independently selected from nitrogen, oxygen, orsulfur, or 4-7 membered heterocyclyl having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur.
 11. Thecompound according to claim 10, wherein n is an integer from 1 to 5,inclusive.
 12. The compound according to claim 10, wherein: L is abivalent C₂₋₈ straight or branched, hydrocarbon chain wherein L has atleast one double bond and at least one methylene unit of L is replacedby —C(O)—, —NRC(O)—, —C(O)NR—, —N(R)SO₂—, —SO₂N(R)—, —S—, —S(O)—, —SO₂—,—OC(O)—, or —C(O)O—, and one or two additional methylene units of L areoptionally and independently replaced by cyclopropylene, —O—, —N(R)—, or—C(O)—; and Y is hydrogen or C₁₋₆ aliphatic optionally substituted withoxo, halogen, NO₂, or CN.
 13. The compound according to claim 12,wherein L is a bivalent C₂₋₈ straight or branched, hydrocarbon chainwherein L has at least one double bond and at least one methylene unitof L is replaced by —C(O)—, and one or two additional methylene units ofL are optionally and independently replaced by cyclopropylene, —O—,—N(R)—, or —C(O)—.
 14. A compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein: one of R^(a) andR^(b) is hydrogen and the other is —OH or —OC(O)R′, or R^(a) and R^(b)are taken together to form an oxo group; R′ is an optionally substitutedgroup selected from C₁₋₆ aliphatic, C₃₋₇ cycloalkyl, 6-10 membered aryl,5-10 membered heteroaryl having 1-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur, or 4-7 membered heterocyclyl having1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur;R^(x) and R^(y) are taken together to form an optionally substitutedC₃₋₇ membered ring having 0-2 heteroatoms independently selected fromnitrogen, oxygen, or sulfur; R¹ is an optionally substituted groupselected from C₁₋₆ aliphatic or C₃₋₇ cycloalkyl(C₁₋₃ alkyl); R² ishydrogen or an optionally substituted group selected from C₁₋₆ aliphaticor C₃₋₇ cycloalkyl; R³ is a warhead —(CH₂)_(n)-L-Y, wherein: n is aninteger from 0 to 5, inclusive; L is a covalent bond, —C(O)—,—N(R)C(O)—, —NH—, —CH₂NH—, —NHCH₂—NHC(O)CH₂OC(O)—, —CH₂NHC(O)—, —NHSO₂—,—NHSO₂CH₂—, —NHC(O)CH₂OC(O)—, or —SO₂NH—, or a bivalent C₁₋₈ saturatedor unsaturated, straight or branched, hydrocarbon chain; and Y isselected from the following (i), (ii), (iii), (iv), (v), (vi), (vii),(viii), (ix), (x), (xi), (xii), (xiii), (xiv), (xv), (xvi), or (xvii):(i) C₁₋₆ alkyl substituted with oxo, halogen, NO₂, or CN; (ii) C₂₋₆alkenyl optionally substituted with oxo, halogen, NO₂, or CN; or (iii)C₂₋₆ alkynyl optionally substituted with oxo, halogen, NO₂, or CN; or(vii) a saturated 3-6 membered carbocyclic ring, wherein said ring issubstituted with 1-4 R^(e) groups; or (ix) a partially unsaturated 3-6membered carbocyclic ring, wherein said ring is substituted with 1-4R^(e) groups; or

(x) (xvii) an 8-10 membered bicyclic, saturated or partially unsaturatedring, wherein said ring is substituted with 1-4 R^(e) groups; wherein:each R is hydrogen or an optionally substituted group selected from C₁₋₆aliphatic, C₃₋₇ cycloalkyl, 6-10 membered aryl, 5-10 membered heteroarylhaving 1-4 heteroatoms independently selected from nitrogen, oxygen, orsulfur, or 4-7 membered heterocyclyl having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur; each R^(e) isindependently selected from -Q-Z, oxo, NO₂, halogen, CN, a suitableleaving group selected from alkoxy, sulphonyloxy, optionally substitutedalkylsulphonyloxy, optionally substituted alkenylsulfonyloxy, optionallysubstituted arylsulfonyloxy, acyl, diazonium, or C₁₋₆ aliphaticoptionally substituted with oxo, halogen, NO₂, or CN, wherein: Q is acovalent bond or a bivalent C₁₋₆ saturated or unsaturated, straight orbranched, hydrocarbon chain, wherein one or two methylene units of Q areindependently replaced by —N(R)—, —S—, —O—, —C(O)—, —OC(O)—, —C(O)O—,—SO—, or —SO₂—, —N(R)C(O)—, —C(O)N(R)—, —N(R)SO₂—, or —SO₂N(R)—; and Zis hydrogen or C₁₋₆ aliphatic optionally substituted with oxo, halogen,NO₂, or CN;

R⁴ is —NHC(O)NHR⁵, —NHC(O)OR⁶, or R⁵ is an optionally substituted groupselected from C₁₋₆ aliphatic, bridged bicyclic, 6-10 membered aryl, 5-10membered heteroaryl having 1-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, or 4-7 membered heterocyclyl having 1-2heteroatoms independently selected from nitrogen, oxygen, or sulfur; R⁶is an optionally substituted group selected from C₁₋₆ aliphatic, bridgedbicyclic, 6-10 membered aryl, 5-10 membered heteroaryl having 1-4heteroatoms independently selected from nitrogen, oxygen, or sulfur, or4-7 membered heterocyclyl having 1-2 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur; R⁷ is an optionally substituted groupselected from C₁₋₆ aliphatic, bridged bicyclic, 6-10 membered aryl, 5-10membered heteroaryl having 1-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, or 4-7 membered heterocyclyl having 1-2heteroatoms independently selected from nitrogen, oxygen, or sulfur; andR is hydrogen or an optionally substituted group selected from C₁₋₆aliphatic, C₃₋₇ cycloalkyl, 6-10 membered aryl, 5-10 membered heteroarylhaving 1-4 heteroatoms independently selected from nitrogen, oxygen, orsulfur, or 4-7 membered heterocyclyl having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur.
 15. Thecompound according to claim 1, wherein R³ is selected from:


16. The compound according to claim 1 selected from the group consistingof:

or a pharmaceutically acceptable salt thereof.
 17. A method forinhibiting HCV protease, or a mutant thereof, activity in a patientcomprising the step of administering to said patient a compoundaccording to claim 1 or a composition thereof.
 18. A compositioncomprising a compound according to claim 1, and a pharmaceuticallyacceptable adjuvant, carrier, or vehicle.
 19. The composition accordingto claim 18, in combination with an additional therapeutic agent. 20.The composition according to claim 19, wherein the additionaltherapeutic agent is an antiviral agent.
 21. A composition comprising acompound according to claim 10, and a pharmaceutically acceptableadjuvant, carrier, or vehicle.
 22. The composition according to claim21, in combination with an additional therapeutic agent.
 23. Thecomposition according to claim 22, wherein the additional therapeuticagent is an antiviral agent.
 24. A composition comprising a compoundaccording to claim 14, and a pharmaceutically acceptable adjuvant,carrier, or vehicle.
 25. The composition according to claim 24, incombination with an additional therapeutic agent.
 26. The compositionaccording to claim 25, wherein the additional therapeutic agent is anantiviral agent.
 27. A composition comprising a compound according toclaim 16, and a pharmaceutically acceptable adjuvant, carrier, orvehicle.
 28. The composition according to claim 27, in combination withan additional therapeutic agent.
 29. The composition according to claim28, wherein the additional therapeutic agent is an antiviral agent.